Use of microbial communities for human and animal health

ABSTRACT

The disclosure relates to a mixture of bacteria belonging to at least six or seven different and specific bacterial species preferably for use in preventing or treating gastro-intestinal disorders. Preferably, the mixture of bacteria is grown together in a fermenter prior to administering the mixture to a subject in order to prevent or treat the disorder.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of co-pending U.S. patent applicationSer. No. 16/068,470, filed Jul. 6, 2018, now U.S. patent ______, whichis a national phase entry under 35 U.S.C. § 371 of International PatentApplication PCT/EP2017/052422, filed Feb. 3, 2017, designating theUnited States of America and published as International PatentPublication WO 2017/134240 A1 on Aug. 10, 2017, which claims the benefitunder Article 8 of the Patent Cooperation Treaty to European PatentApplication Serial No. EP16154288.1, filed Feb. 4, 2016, the contents ofthe entirety of each of which are incorporated herein by this reference.

TECHNICAL FIELD

This application relates to a mixture of bacteria belonging to at leastsix or seven different and specific bacterial species, preferably foruse to prevent or treat gastro-intestinal disorders. More preferably,the mixture of bacteria is grown together in a fermenter prior toadministering the mixture to a subject in order to prevent or treat thedisorder.

STATEMENT ACCORDING TO 37 C.F.R. § 1.821(c) or (e)-SEQUENCE LISTINGSUBMITTED AS ASCII TEXT FILE

Pursuant to 37 C.F.R. § 1.821(c) or (e), a file containing an ASCII textversion of the Sequence Listing has been submitted concomitant with thisapplication, the contents of which are hereby incorporated by reference.

BACKGROUND

The human and animal gut ecosystem consists of a variety of differenthabitats and metabolic niches that are colonized by the so-calledmicrobiota that contain more than 10¹¹ micro-organisms per gram wetweight of contents, predominantly anaerobes (Macfarlane & Macfarlane,1997). It is nowadays well-recognized that the human or animal gutmicrobiome plays a crucial role in human health and well-being bycontributing to energy harvest, modulating the immune system andestablishing colonization resistance against opportunistic pathogens(Fuller & Gibson, 1997; Cummings & Macfarlane, 1997). Evidence existsthat the interaction of bacteria and their metabolites with the mucuslayer and/or with the intestinal gut wall is important (Barnett et al.2012). Although the gut microbiome is generally stable over time, itscomposition is altered by external perturbations, such as dietarychanges, antibiotic use, increasing hygienization and stress. This leadsto an unbalanced condition in the gastrointestinal tract, calleddysbiosis (Clemente et al. 2012). Dysbiosis is characterized by moderateor severe disruptions in the normal gut microbiome composition, therebycausing the lack of key microbial species, gaps in specific microbialfunctions and, as a consequence, an impaired modulation of the gut wallactivity. This may lead to the colonization of pathogenicmicroorganisms—causing diarrhea or necrotizing enteritis (Sekirov et al.2008). One of the extreme forms of such pathogenesis is CDAD(Clostridium difficile associated diarrhea) for which classic antibiotictherapy is more and more falling short of curing the patient. Otherconsequences of microbial dysbiosis may be a compromised immuneresponse, resulting in chronic inflammation (Willing et al. 2009) orfood allergies, or an increased gut permeability, nutrient malabsorptionor even bacteremia. The adverse effects of dysbiosis toward microbialfunctionality and gut wall physiology may thus undermine human health.In fact, constipation, IBS, IBD, pouchitis, metabolic syndrome, obesity,diabetes, cardiovascular diseases, mental conditions, impaired cognitivefunction, a neurodegenerative disease, different types of cancers (e.g.,colon cancer), inflammation of the female reproductive apparatus, CDAD,rheumatism or rheumatoid arthritis are all associated with changes inthe activity/composition of the gut microbiota. It is, therefore, clearthat dysbiosis should be avoided or remedied upon occurrence.

When dysbiosis is associated with the presence of pathogens, an obviousstrategy to get rid of health-detrimental microorganisms is theapplication of antibiotic agents. However, widespread and improper useof broad-spectrum antibiotics over the last decades has dramaticallyincreased antibiotic resistance (Brandi et al. 2008). Moreover,antibiotics also tackle the indigenous gut microorganisms, many of whichfulfill crucial functions and deliver health benefits, thereforeworsening the condition of dysbiosis. As a result, the last two decadeshave seen a tremendous increase in functional food research,particularly the development of prebiotic and probiotic products.Although the prebiotic concept is attractive as it concerns the dietarymodulation of indigenous gut microorganisms that are already adapted tothe host (Van Loo et al. 1999), it is primarily used in a preventivemanner. For a therapeutic application, a severely disrupted gutmicrobiome would benefit more from the introduction of key microbialspecies, rather than the provision of substrates that benefithealth-promoting species that are less abundant or even absent in adiseased individual. A possible solution is the introduction of viable,health-promoting microorganisms, termed probiotics (Iannitti andPalmieri, 2010). Probiotic products are mostly comprised of one to acouple of not interconnected microbial strains (mostly lacticacid-producing bacteria) with a specific functionality. However,survival of probiotic strains during the harsh conditions of the upperdigestive tract is challenging and competition with the vast indigenousmicrobiome is often negligible. Yet, the concept of introducing newspecies in a compromised gut ecosystem has gained momentum in recentyears through the application of fecal microbial transplants (FMT)(Khoruts et al. 2010). This entails the transfer of a fecal microbialslurry from a healthy donor to a diseased recipient. This form ofbacteriotherapy is mostly applied to treat antibiotic-resistantinfections and has cure rates of 90% and higher. FMT is currently beingconsidered for treating many other pathologies that have their origin ingastrointestinal dysbiosis (Crohn's disease, obesity, irritable bowelsyndrome, etc.). FMT seems to efficiently work where single probioticstrains frequently fail. Yet, the badly characterized nature of fecaltransplants comes with transmission risks of infectious diseases andcurrently raises questions over its widespread applicability in lessacute and life-threatening pathologies (De Vrieze 2013).

Early 2013, an alternative for fecal microbial transplants entered thefield with the publication of a scientific paper (Petrof et al. 2013)and patent application (WO 2013/037068—Method for treatment of disordersof the gastrointestinal system) on the use of a synthetic mixture ofmicrobes that were isolated from an individual based on theirculturability as therapeutic agent to cure CDAD. Such product is alsocomposed of a known set of microorganisms, which would take away theconcerns of disease transmission from fecal transplants, when QPScriteria are respected. However, mixing together microorganisms does notguarantee them to interact with one another and to occupy functionalniches that require microbial networking. Product stability,standardization and performance of important functions can, therefore,not be guaranteed.

In the patent application WO 2014/145958A2 (Network-based microbialcompositions and methods), it is proposed to administer to a mammaliansubject, in need of an effective amount of a therapeutic bacterialcomposition, a plurality of isolated bacteria or a purified bacterialpreparation. The plurality of isolated bacteria or the purifiedbacterial preparation is able to form a so-called network ecology. Thebacteria belonging to this preparation are selected based on genomicinformation and are provided to the mammalian subject as a looselyassembled set of strains.

In a publication of Becker et al. (2011), a community is describedconsisting of eight different strains: Anaerostipes caccae, Bacteroidesthetaiotaomicron, Bifidobacterium longum, Blautia producta, Clostridiumbutyricum, Clostridium ramosum, Escherichia coli, and Lactobacillusplantarum. The community is referred to as SIHUMIx (Simplified HumanMicrobiota extended). This artificial microbial community was tested inrat studies, comparing SIHUMIx-inoculated rats with conventionalhuman-associated and germ-free rats. The authors claim the community isrepresentative for the human colon-associated microbiota in terms ofcomposition and functionality. The microbial community evolved dependingon the age of the rats, but reached a stable composition over time.

Van den Abbeele et al. (2013) suggested the possibility of creating aglycan-degrading community by using conventional in vitro fermentersthat can be inoculated with relevant keystone species and a mixture ofcross-feeding microbes. After inoculation and stabilization, such amicrobial network unit for specific functions can be attained andproduced at a large scale.

Finally, Newton et al. (1998) made use of anaerobic chemostats to createreproducible defined bacterial communities comprising fourteen differentsaccharolytic and amino acid-fermenting species (i.e., Bifidobacteriumlongum, Bif. adolescentis, Bif. pseudolongum, Bif. infantis, Bacteroidesthetaiotaomicron, Bact. vulgatus, Lactobacillus acidophilus,Enterococcus faecalis, Ent. faecium, Escherichia coli, Clostridiumperfringens, Cl. butyricum, Cl. innocuum, Cl. Bifermentans) to study theeffect of the sulphate-reducing bacterium (SRB) Desulfovibriodesulfuricans on other intestinal organisms.

However, there is still a need to design alternative and specificmixtures of bacterial species that can be effectively used to prevent ortreat gastro-intestinal disorders. Moreover, it is completely unknownwhether pre-adapted mixtures perform therapeutically as well, worse orbetter when compared to administering the loosely assembled andnon-pre-adapted mixtures of the same bacterial species.

BRIEF SUMMARY

This disclosure relates in the first instance to a compositionconsisting essentially of bacteria belonging to the speciesFaecalibacterium prausnitzii, Butyricicoccus pullicaecorum, Roseburiainulinivorans, Roseburia hominis, Akkermansia muciniphila, Lactobacillusplantarum and Anaerostipes caccae preferably for use to prevent or treatsymptoms associated with a gastro-intestinal disorder.

In other words, the disclosure relates to a method of preventing ortreating symptoms associated with a gastro-intestinal disorder in asubject in need thereof, the method comprising administering to thesubject a therapeutically effective amount of a composition consistingessentially of bacteria belonging to the species Faecalibacteriumprausnitzii, Butyricicoccus pullicaecorum, Roseburia inulinivorans,Roseburia hominis, Akkermansia muciniphila, Lactobacillus plantarum andAnaerostipes caccae.

This disclosure further relates to a composition as described whereinthe gastro-intestinal disorder is a disruption of the barrier functionof the gut, diarrhea, constipation, irritable bowel syndrome,inflammatory bowel disease, Crohn's disease, ulcerative colitis, coeliacdisease, pouchitis, mucositis, an infection of the gut, gut microbiotadysbiosis, or any combination thereof.

Also disclosed is a composition as described herein, wherein thegastro-intestinal disorder is prevented or treated via: a) stimulatinggrowth and/or activity of one or a limited number of beneficial bacteriain the intestinal tract, b) inhibiting growth and/or activity of one ora limited number of pathogenic bacteria in the intestinal tract, c)relatively increasing the attachment of non-pathogenic bacteria to themucosa of the gastrointestinal surface, d) reducing uncontrolled uptakeof antigens, pro-inflammatory, bacteria or bacterial products by thegut, e) providing anti-inflammatory activity at the intestinal surface,f) increasing gut barrier functioning, g) producing bacterialmetabolites or h) any combination of a) to g).

The disclosure also relates to a composition as described herein whereinbacteria belonging to the species Roseburia hominis are eliminated fromthe composition.

The disclosure further relates to a composition as described hereinwherein bacteria belonging to the species Escherichia coli, Enterococcusfaecium, Lactobacillus mucosae, Bifidobacterium adolescentis,Bifidobacterium longum, Bacteroides thetaiotaomicron and Bacteroidesvulgatus are further added to the composition.

The disclosure further relates to a composition as described hereinfurther comprising one or more prebiotics.

In a preferred embodiment, this disclosure relates to a composition asdescribed herein wherein the bacteria are preadapted by growing themtogether in a fermenter prior to administering the composition to thesubject to prevent or treat the gastro-intestinal disorders.

In this regard, the disclosure further relates to a composition asdescribed herein wherein the fermenter is a dynamic simulator of thegastro-intestinal tract.

More specifically, this disclosure relates to a composition as describedherein wherein the bacteria are chosen from the list of the followingstrains: Faecalibacterium prausnitzii LMG P-29362, Faecalibacteriumprausnitzii DSMZ 17677, Butyricicoccus pullicaecorum LMG P-29360,Butyricicoccus pullicaecorum LMG 24109, Roseburia inulinivorans LMGP-29365, Roseburia inulinivorans DSMZ 16841, Roseburia hominis LMGP-29364, Roseburia hominis DSMZ 16839, Akkermansia muciniphila LMGP-29361, Akkermansia muciniphila DSMZ 22959, Lactobacillus plantarum LMGP-29366, Lactobacillus plantarum ZJ316, Anaerostipes caccae LMG P-29359,Anaerostipes caccae DSMZ 14662 and/or strains showing at least 97%sequence identity to the 16SrRNA sequences of at least one of thestrains.

The disclosure further relates to a composition as described hereinwherein the composition is a pharmaceutical composition formulatedeither as a rectally administrated form or an orally ingestible form.

In this regard, the disclosure further relates to a composition asdescribed herein wherein the orally ingestible form is a capsule,microcapsule, tablet, granule, powder, troche, pill, suspension orsyrup.

The disclosure further relates to a composition as described herein thatis incorporated in a food, drink, food supplement or nutraceutical.

This disclosure more specifically relates to a composition as describedherein wherein the composition comprises between 10⁵ and 10¹¹colony-forming units of bacteria.

The gut microbiome comprises hundreds of microbial species that co-existwithin different subjects and that interact with each other and thehost. Nowadays, it is generally believed that the gut microbiota play akey role in human health and disease by regulating metabolic functionsand immune homeostasis (Cent et al. 2014). Several studies haveinvestigated these complex gut microbial communities in an attempt todefine a “core microbiome,” implying that all human individuals share akey number of essential species or strains that define the functionalcapabilities of a healthy gut microbiome (Kinross et al., 2011). Basedon this concept (i.e., that all humans are populated by a coremicrobiome), the extensive literature that is available on thecomposition and function of the gut microbiota (e.g., keystone species,mucosal versus luminal microbiota, proximal versus distal colonbacteria, etc.) and functional genome analysis, a list of microbialcandidates could be identified that covers the main functionalities ofthe complex human gut microbiome.

The disclosure relates in the first instance to a specific selection ofa subgroup of bacterial species of the human gut microbiome that have aparticular and surprising effect. More specifically, the disclosurerelates to a composition consisting essentially of bacteria belonging tothe species Faecalibacterium prausnitzii, Butyricicoccus pullicaecorum,Roseburia inulinivorans, Roseburia hominis, Akkermansia muciniphila,Lactobacillus plantarum and Anaerostipes caccae preferably for use toprevent or treat symptoms associated with a gastro-intestinal disorder.The term “consisting essentially of” indicates that the composition mayinclude other bacterial species and/or other components provided they donot negatively affect the effect (i.e., preventing or treating symptomsassociated with a gastro-intestinal disorder) of the composition. In anembodiment, a composition of the disclosure comprises bacteria belongingto the species Faecalibacterium prausnitzii, Butyricicoccuspullicaecorum, Roseburia inulinivorans, Roseburia hominis, Akkermansiamuciniphila, Lactobacillus plantarum and Anaerostipes caccae.

In another embodiment, a composition of the disclosure consists ofbacteria belonging to the species Faecalibacterium prausnitzii,Butyricicoccus pullicaecorum, Roseburia inulinivorans, Roseburiahominis, Akkermansia muciniphila, Lactobacillus plantarum andAnaerostipes caccae.

The bacterial species Faecalibacterium prausnitzii (Duncan et al. 2002),Butyricicoccus pullicaecorum (Eeckhaut et al. 2008), Roseburiainulinivorans (Duncan et al. 2006), Roseburia hominis (Duncan et al.2006), Akkermansia muciniphila (Derrien et al. 2004), Lactobacillusplantarum (Walter 2008) and Anaerostipes caccae (Schwiertz et al. 2002)are well-known bacterial species to a skilled person. The terms“symptoms associated with a gastro-intestinal disorder” refer to healthproblems in humans and animals. The use of a composition of thedisclosure leads more specifically to prevention/recovery from dysbiosisresulting in a positive modulation of the interaction between bacteriaand intestinal surface. As a result, an improved functioning of theintestinal surface is obtained: e.g., barrier, hormonal, immunefunctioning. The onset of the effect on the intestinal surface isquicker when a “pre-adapted composition” is dosed as compared to a“loosely assembled set of the same strains” (see further). As usedherein, modulating or improving the barrier, hormonal or immune functionof the intestinal surface is meant to include altering any parameterthat affects the normal homeostasis of the intestinal surface and, inparticular, its role in the first line of defense against the invasionby pathogens, antigens or other harmful substances and its role toproduce substances (e.g., immune molecules, hormones) that have systemicinfluences on the host. The parameters include, but are not limited to:

-   -   a stimulation of the growth and/or activity of one or a limited        number of beneficial bacteria in the intestinal tract (e.g.,        lactobacilli, bifidobacteria, butyrate- or propionate-producing        bacteria, others);    -   an inhibition of the growth and/or activity of one or a number        of pathogenic bacteria in the intestinal tract;    -   a relative increase in the attachment of non-pathogenic bacteria        to the mucosa of the intestinal surface;    -   a reduction in the uncontrolled uptake from the gut of antigens,        pro-inflammatory molecules, bacteria or bacterial products;    -   modulation of the gut-associated lymphoid tissue (GALT) and the        host systemic immune system;    -   production of specific bacterial metabolites (e.g., propionate,        butyrate); and    -   modulation of the production of certain intestinal signaling        molecules that directly or indirectly modulate metabolic        homeostasis (e.g., pro-glucagon, GLP-1, GLP-2, FIAF).

This disclosure thus relates to a composition as described hereinwherein the gastro-intestinal disorder is prevented or treated via: a)stimulating growth and/or activity of one or a limited number ofbeneficial bacteria in the intestinal tract, b) inhibiting growth and/oractivity of one or a limited number of pathogenic bacteria in theintestinal tract, c) relatively increasing the attachment ofnon-pathogenic bacteria to the mucosa of the gastrointestinal surface,d) reducing uncontrolled uptake of antigens, pro-inflammatory, bacteriaor bacterial products by the gut, e) providing anti-inflammatoryactivity at the intestinal surface, f) increasing gut barrierfunctioning, g) producing bacterial metabolites or h) any combination ofa) to g).

Health conditions that may be associated with general gastro-intestinaldisorders include, but are not limited to, constipation, Irritable BowelSyndrome (IBS), Inflammatory Bowel Diseases (IBD), gut microbiotadysbiosis, mucositis, metabolic syndrome, obesity, diabetes, acardiovascular disease, chronic fatigue syndrome, a mental condition,impaired cognitive function, a neurodegenerative disease, a form ofcancer, an autoimmune condition, impaired immune functioning,rheumatism, rheumatoid arthritis, inflammation of the femalereproductive apparatus, and infection of pathogens (bacteria, virusesand fungi). Examples of neurodegenerative diseases include, but are notlimited to, ALS, dementia, Alzheimer's, Parkinson's and Huntington'sdisease. Examples of types of cancers include, but are not limited to,lung cancer, breast cancer, prostate cancer, pancreatic cancer andparticularly colorectal cancer. Examples of autoimmune diseases include,but are not limited to, multiple sclerosis, atopic dermatitis, celiacdisease, psoriasis and lupus.

Based on the observation that the compositions of the disclosure enhancethe interaction and/or activity of non-pathogenic bacteria to themucosal layer of the gastrointestinal epithelium, it is envisaged thatthe preparations are particularly useful to improve the barrier functionof the intestinal surface, such as, for example, to prevent or reducethe uncontrolled uptake from the gut of antigens, pro-inflammatorymolecules, pathogenic bacteria or bacterial products. One suchindication with an impaired mucosal barrier is Inflammatory BowelDisease. As it is generally accepted that in Inflammatory BowelDiseases, mucosal injury with an impaired resolution of the lesions isone of the key elements that lead to these chronic indications, thecompositions of the disclosure have a beneficial effect in thatindication. Provided is the use of the compositions of the disclosure inthe prevention and treatment of conditions associated with an impairedbarrier function and characterized by the uncontrolled uptake from thegut of antigens, pro-inflammatory molecules, pathogenic bacteria orbacterial products.

“Inflammatory bowel diseases,” also referred to as “chronic colonicdiseases,” as used herein include any condition characterized bypersistent mucosal inflammation at different levels of thegastrointestinal tract, such as, for example, inflammatory bowelsyndrome, mucositis, gastric ulcers, Crohn's disease, ulcerativecolitis, colorectal cancer and pouchitis.

As mucositis is generally recognized as being essentially characterizedby inflammation of the mucosal surface lining the mouth andgastrointestinal tract, typically as adverse event of chemotherapy andradiotherapy or stem cell transplantation, it is also to be envisagedthat the application of the compositions of the disclosure have abeneficial effect in that indication. Thus, also provided herein is theuse of the compositions of the disclosure in the prevention andtreatment of conditions associated with mucositis. Mucositis can occuranywhere along the gastrointestinal tract. In the case of occurring inthe oral cavity, it is typically referred to as oral mucositis.

It is also to be envisaged that the application of the compositions ofthe disclosure provide protection against introduction of antigens thatcause allergic reactions, whereby such allergens may comprise certainfood substances, chemicals and other molecules. Thus, in a furtherembodiment, provided is the use of compositions in the prevention andtreatment of conditions associated with the introduction of antigensleading to allergic reactions (e.g., food allergies, asthma, andeczema).

It is furthermore also envisaged that the application of thecompositions to influence both the gut-associated lymphoid tissue (GALT)as well as the systemic immune system. Among other effects, this mayresult in decreased expression of pro-inflammatory cytokines andincreased production of immunoregulatory factors and improved activityof lymphocytes. It is, therefore, envisaged that the compositions beparticularly useful in improving the development and functioning of thehost immune system.

In another aspect of the disclosure, based on the observation that thecompositions of the disclosure modulate the epithelial barrier andsubsequently decrease chronic inflammation, it is envisaged that thecompositions be particularly useful in controlling and improvingmetabolic homeostasis. Non-limiting effects of the preparations onmetabolic homeostasis include control of food intake and fat and glucosemetabolism, improvement of insulin secretion and sensitivity and controlof cholesterol synthesis and metabolism. Thus, also provided herein isthe use of the compositions of the disclosure in the management of fooduptake, induction of satiety, weight management, and the prevention andtreatment of conditions associated with an impaired metabolichomeostasis, such as obesity and type 2 diabetes.

Based on the observation that a composition of the disclosure decreasesseveral established causal risk factors of cardiovascular diseases(CVD), it is to be envisaged in another aspect of the disclosure thatthe compositions be particularly useful for the prevention of CVD. CVDtechnically refers to any disease that affects the cardiovascularsystem, yet is usually used to refer to those related toatherosclerosis. The latter is a syndrome affecting arterial bloodvessels, a chronic inflammatory response in the walls of arteries, inlarge part due to the accumulation of macrophage white blood cells andpromoted by low density lipoproteins. CVD development depends onmultiple mechanisms and a number of clear causal risk factors have beenidentified. These factors include, yet are not limited to, elevated LDLcholesterol, plasma triglycerides, metabolic diseases (obesity,diabetes, etc.), chronic inflammation and oxidative stress. The lattertwo factors are especially of utmost importance. Atherosclerosisdevelops from LDL becoming oxidized (LDL-ox) by free radicals,particularly oxygen free radicals, in situations of oxidative stress.Excessive response of the immune system, in case of chronicinflammation, to damage caused by LDL-ox further promotes the expansionof the disease. Thus, also provided herein is the use of thecompositions of the disclosure in the prevention or treatment of CVD.

In a further aspect, given the beneficial effect of the compositions ofthe disclosure on the adherence of the normal microbiota to the mucosallayer, it is envisaged that the application of the compositions providesprotection against mucosal attachment and invasion by pathogens.Examples of pathogens include, but are not limited to, Bacillusanthracis, Bacillus cereus, Bordetella pertussis, Borrelia burgdorferi,Brucella abortus, Brucella canis, Brucella melitensis, Brucella suis,Campylobacter jejuni, Chlamydia pneumonia, Chlamydia trachomatis,Chlamydophila psittaci, Clostridium botulinum, Clostridium difficile,Clostridium perfringens, Clostridium tetani, Corynebacterium diphtheria,Enterotoxigenic Escherichia coli (ETEC), Enteropathogenic E. coli, E.coli O157:H7, Francisella tularensis, Haemophilus influenza,Helicobacter pylori, Legionella pneumophila, Leptospira interrogans,Listeria monocytogenes, Mycobacterium leprae, Mycobacteriumtuberculosis, Mycoplasma pneumonia, Neisseria gonorrhoeae, Neisseriameningitides, Pseudomonas aeruginosa, Rickettsia rickettsii, Salmonellatyphi, Salmonella typhimurium, Shigella sonnei, Staphylococcus aureus,Staphylococcus epidermidis, Staphylococcus saprophyticus, Streptococcusagalactiae, Streptococcus pneumonia, Streptococcus pyogenes, Treponemapallidum, Vibrio cholera, Yersinia pestis, Candida spp., Norovirus(Norwalk Virus), and Hepatitis A, and viruses inducing smallpox,influenza, mumps, measles, chickenpox, ebola, and rubella. Thus, in afurther embodiment, the disclosure provides the use of the compositionsof the disclosure in the prevention and treatment of conditionsassociated with the mucosal attachment and invasion by pathogens, inparticular, in the treatment and prevention of acquired diarrhea andtraveler's diarrhea.

The disclosure thus relates to a method to prevent or treat symptomsassociated with a gastro-intestinal disorder in a subject in needthereof comprising administering a therapeutically effective amount of acomposition consisting essentially of bacteria belonging to the speciesFaecalibacterium prausnitzii, Butyricicoccus pullicaecorum, Roseburiainulinivorans, Roseburia hominis, Akkermansia muciniphila, Lactobacillusplantarum and Anaerostipes caccae.

The term “subject in need” refers to a human or a non-human animalhaving a gastro-intestinal disorder as described herein.

The terms “a therapeutically effective amount” refers to a minimum ofthe combined total amount of the seven bacterial species that is capableof exerting its prophylactic or therapeutic effect. The seven bacteriaspecies are Faecalibacterium prausnitzii, Butyricicoccus pullicaecorum,Roseburia inulinivorans, Roseburia hominis, Akkermansia muciniphila,Lactobacillus plantarum and Anaerostipes caccae.

However, “a therapeutically effective amount” may also refer to aminimum of a combined total amount of six bacteria species:Faecalibacterium prausnitzii, Butyricicoccus pullicaecorum, Roseburiainulinivorans, Akkermansia muciniphila, Lactobacillus plantarum andAnaerostipes caccae.

Depending on the final application, the combined total amount can be theresult of equal amounts of each of the seven bacterial species orunequal amounts of the seven bacterial species, in which each singlespecies of the seven bacterial species has a minimum abundance of0.0001% of the combined total amount, more preferably a minimumabundance of 0.001% of the combined total amount and most preferably aminimum abundance of 0.01% of the combined total amount. If thus, forexample, six species have an abundance of 10.00% of the combined totalamount, then the seventh species has an abundance of 40.00% of thecombined total amount. Depending on the final application, the combinedtotal amount ranges between a daily dose of 10² and 10¹⁴ bacterialcells, preferably ranging between a daily dose of 10³ and 10¹³ bacterialcells, more preferably ranging between a daily dose of 10⁴ and 10¹²bacterial cells and most preferably ranging between a daily dose of 10⁵and 10¹¹ bacterial cells.

The disclosure further relates to a composition as described hereinwherein bacteria belonging to the species Roseburia hominis areeliminated from the composition. The term “eliminated” refers, inparticular, to making a composition of six bacterial species as isindicated further in the Examples section without adding or removing thespecies Roseburia hominis as a seventh species.

The disclosure further relates to a composition as described hereinwherein bacteria belonging to the species Escherichia coli, Enterococcusfaecium, Lactobacillus mucosae, Bifidobacterium adolescentis,Bifidobacterium longum, Bacteroides thetaiotaomicron and Bacteroidesvulgatus are further added to the composition.

The bacterial species Escherichia coli (Rath et al. 1999), Enterococcusfaecium (Schleifer et al. 1984), Lactobacillus mucosae (Roos et al.2000), Bifidobacterium adolescentis (Scharek et al. 2000),Bifidobacterium longum (Bahaka et al. 1993), Bacteroidesthetaiotaomicron (Scharek et al. 2000) and Bacteroides vulgatus (Rath etal. 1999) are well known bacterial species to a skilled person. Thedisclosure further relates to a composition as described herein furthercomprising one or more prebiotics.

The term “prebiotic” refers to any chemical that induces the growth oractivity of microorganisms (e.g., bacteria) that contribute to thewell-being of their host. Hence, prebiotics can influence or alter thecomposition of organisms in the gut microbiome. However, in principle,it is a more general term that can refer to other areas of the body aswell. Typical, but non-limiting, prebiotics are non-digestible fibercompounds that at least partially pass undigested through the upper partof the gastrointestinal tract and stimulate the growth or activity ofadvantageous bacteria that colonize the large bowel by acting assubstrate for them.

In a preferred embodiment, the disclosure relates to a composition asdescribed herein wherein the bacteria are grown together in a fermenterprior to administering the composition to prevent or treat thegastro-intestinal disorders. The latter compositions are also referredto (see further) as the “Collaborome strategy” or as the “Alternativecollaborome strategy.” In contrast, compositions wherein the bacteriaare not grown together in a fermenter prior to administration arereferred to (see further) as the “Assembly strategy.”

In this regard, this disclosure further relates to a composition asdescribed herein wherein the fermenter is a dynamic simulator of thegastro-intestinal tract. In this specific case, the latter compositionsare also referred to (see further) as the “Collaborome strategy.”

The SHIME® (Simulator of the Human Microbial Ecosystem) is a dynamic invitro model of the human gastrointestinal tract that is composed of fivedouble jacketed vessels, simulating the stomach, small intestine, andthe three colon regions (ascending, transverse, and descending colon),with a total retention time of 72 hours (FIG. 1). Three times per day,140 ml SHIME® feed and 60 ml pancreatic juice were added to the stomachand small intestine compartments, respectively (Van den Abbeele et al.,2010). After an initial two-week stabilization period, which allows themicrobiota to adapt to the imposed in vitro conditions, the isolationprocedure was started. The selected microbial strains of the disclosurecan thus be inoculated in single-stage (alternative collaboromestrategy) or multi-stage reactors or dynamic simulators of thegastrointestinal tract (e.g., SHIME® or M-SHIME®, collaborome strategy)under standardized conditions representative for the GI tract.Accordingly, the disclosure relates to a reactor comprising acomposition comprising, consisting of or consisting essentially ofbacteria belonging to six or seven or up to fourteen species as definedherein and further listed below:

-   -   comprising a composition comprising, consisting of or consisting        essentially of bacteria belonging to the species        Faecalibacterium prausnitzii, Butyricicoccus pullicaecorum,        Roseburia inulinivorans, Roseburia hominis, Akkermansia        muciniphila, Lactobacillus plantarum, and Anaerostipes caccae,        or    -   comprising a composition comprising, consisting of or consisting        essentially of bacteria belonging to the species        Faecalibacterium prausnitzii, Butyricicoccus pullicaecorum,        Roseburia inulinivorans, Akkermansia muciniphila, Lactobacillus        plantarum, and Anaerostipes caccae or,    -   comprising a composition comprising, consisting of or consisting        essentially of bacteria belonging to the species        Faecalibacterium prausnitzii, Butyricicoccus pullicaecorum,        Roseburia inulinivorans, Roseburia hominis, Akkermansia        muciniphila, Lactobacillus plantarum, Anaerostipes caccae,        Escherichia coli, Enterococcus faecium, Lactobacillus mucosae,        Bifidobacterium adolescentis, Bifidobacterium longum,        Bacteroides thetaiotaomicron, and Bacteroides vulgatus.

In a preferred embodiment, this reactor comprising the composition isunder standardized conditions representative for the GI tract as definedbelow.

The parameters characterizing the standardized conditions include, butare not limited to, pH (ranging between 1.5 and 8); availability ofcarbon sources (either carbohydrate or proteins or a combinationthereof); retention time in a specific reactor (ranging between 10minutes and 200 hours); oxygen availability (ranging between 0 and 8g/L); availability of micronutrients; presence/absence of antibiotics;concentration of bile salts (ranging between 0 and 20 mM); presence ofheavy metals; and presence of host factors as immune molecules. In apreferred embodiment, the parameters characterizing the standardizedconditions comprise pH, retention time in a specific reactor andconcentration of bile salts, all as earlier defined herein. Depending onthe complexity of the collaborome, a period of 1 to 15 days is needed toobtain a functionally stable collaborome. On average, in order todevelop a collaborome composed of 7 to 14 members, a time between 3 and10 days is sufficient to obtain a functionally stable collaborome(depending on the environmental conditions). A composition as definedherein is, therefore, obtainable after having been trained or culturedduring a time between 3 and 10 days under conditions wherein pH,retention time in a specific reactor, and concentration of bile saltshave been set as defined herein. Such a process allows the production ofa composition or collaborome that is functionally stable.

Within the context of the disclosure, “a functionally stablecollaborome” is a composition as defined herein still comprising theinitial different number of species of bacteria after at least 3 or 5 or10 days of culture.

In a further aspect, there is provided a reactor operating understandardized conditions representative for the GI tract, comprising: pHranging between 1.5 and 8; availability of carbon sources; retentiontime between 10 minutes and 200 hours; oxygen availability between 0 and8 g/L; availability of micronutrients; presence/absence of antibiotics;concentration of bile salts between 0 and 20 mM; presence of heavymetals; and presence of host factors as immune molecules. In anembodiment, the reactor is such that the parameters characterizing thestandardized conditions comprise pH, retention time in a specificreactor and concentration of bile salts as defined in the previousparagraph. In an embodiment, such reactor comprises a composition of 5and 20 distinct bacteria members, or 6 to 14 distinct bacteria membersor 5 to 15 distinct bacteria members. In a preferred embodiment, suchcomposition resides for a time between 3 and 14 days or 3 and 10 days insuch a reactor to obtain a functionally stable collaborome.

The disclosure more specifically relates to the composition and use of aset of microbial strains having specific functional characteristics andpre-adapted to function together in order to prevent or treat healthproblems in humans and animals and obtaining a faster biotherapeuticonset and higher efficiency as compared to a loosely assembled set ofthe same strains (=“assembly strategy”). Such a set of microorganismspre-adapted to function together takes the name of the “collaboromestrategy” or “alternative collaborome strategy.”

In other words, the disclosure relates to pre-adapted compositions ofsets of microbial strains preferably for use to significantly decreasethe time of biotherapeutic onset and/or to significantly increase theeffect of treatment of dysbiosis as compared to a loosely assembled setof the same microbial strains.

The terms “significantly decrease the time of biotherapeutic onset” meanthat, by being pre-adapted, the set of microorganisms can exert theirfunctionality at least 5% quicker (on a temporal scale), preferably atleast 10% quicker, more preferably at least 20% quicker and mostpreferably at least 30% quicker as compared to a loosely assembled setof the same strains. Any value below 5% is considered physiologicallynot relevant.

The terms “significantly increase the effect of treatment” mean that, bybeing pre-adapted, the set of microorganisms can exert theirfunctionality with at least a 5% higher efficacy, preferably at least10% more efficient, more preferably at least 20% and most preferably atleast 30% more efficient. The efficacy depends on the endpoint for whichthe set of microorganisms has been designed. Possible functionalitiesinclude, but are not limited to, Short Chain Fatty Acid (SCFA)production, improvement in gut barrier permeability, decrease/increasein pro-inflammatory cytokines, increase in anti-inflammatory cytokines,decrease in pathogen concentration (at least 0.5 log), decrease in gasproduction, and stimulation of specific gut-wall receptors, etc. Anyvalue below 5% is considered physiologically not relevant.

Hence, the disclosure more specifically relates to a method to preventor treat dysbiosis of humans and animals in need thereof comprisingadministering a therapeutic amount of a pre-adapted composition of a setof microbial strains to the humans or animals wherein the treatmentresults in a faster biotherapeutic onset and/or increased efficiency ascompared to the administration of a loosely assembled set of the samemicrobial strains.

More specifically, the disclosure relates to a composition as describedherein wherein the bacteria are chosen from the list of the followingstrains: Faecalibacterium prausnitzii LMG P-29362, Faecalibacteriumprausnitzii DSMZ 17677, Butyricicoccus pullicaecorum LMG P-29360,Butyricicoccus pullicaecorum LMG 24109, Roseburia inulinivorans LMGP-29365, Roseburia inulinivorans DSMZ 16841, Roseburia hominis LMGP-29364, Roseburia hominis DSMZ 16839, Akkermansia muciniphila LMGP-29361, Akkermansia muciniphila DSMZ 22959, Lactobacillus plantarum LMGP-29366, Lactobacillus plantarum ZJ316, Anaerostipes caccae LMG P-29359,Anaerostipes caccae DSMZ 14662 and/or strains showing at least 97%sequence identity to the 16SrRNA sequences of at least one of thestrains.

The above-indicated strains having accession numbers LMG P-29362 (dateof deposit: Jan. 18, 2016), LMG P-29360 (date of deposit: Jan. 18,2016), LMG P-29365 (date of deposit: Jan. 18, 2016), LMG P-29364 (dateof deposit: Jan. 18, 2016), LMG P-29361 (date of deposit: Jan. 18,2016), LMG P-29366 (date of deposit: Jan. 18, 2016) and LMG P-29359(date of deposit: Jan. 18, 2016) have been deposited with BCCM/LMGLaboratorium voor Microbiologie, Universiteit Gent (UGent), having anaddress of K. L. Ledeganckstraat 35, B-9000 Gent, Belgium.

The above-indicated strains having accession numbers DSMZ 17677,LMG24109, DSMZ 16841, DSMZ 16839, DSMZ 22959, ZJ316 and DSMZ 14662 havebeen deposited in public collections, have been described intensivelyand are accessible to skilled persons worldwide.

It should be further clear that variants of each of the strains showingat least 97% (i.e., 97, 98, 99%) sequence homology to the 16S rRNAsequence of each of the corresponding strains are also part of thisdisclosure. An example to determine such sequence “homology” is, forinstance, described by Eeckhaut et al. (2008). As used herein, the term“16S rRNA” refers to a nucleic acid sequence of about 1542 nucleotides,which is a component of the small prokaryotic ribosomal subunit (30S).The 16S rRNA is known to act as a scaffold defining the positions of theribosomal proteins. The 16S rRNA sequence is commonly used forphylogenetic studies, as it is known to be a highly conserved sequence.Comparative analysis of 16S rRNA sequences from thousands of organismshas demonstrated the presence of oligonucleotide signature sequences. Asused herein, the term “homology” refers to the sequence similarity ofthe nucleic acids. For example, in general, if two nucleic acids haveidentical sequences they show 100% homology. A change in the nucleotidesequence of one of the nucleic acids reduces the percentage of homology.In general, the percentage homology quantifies the degree of identitybetween two nucleic acid sentences.

Sequence identity or sequence homology is herein defined as arelationship between two or more amino acid (polypeptide or protein)sequences or two or more nucleic acid (polynucleotide) sequences, asdetermined by comparing the sequences. Usually, sequence identities orsimilarities are compared over the whole length of the sequencescompared. In the art, “identity” also means the degree of sequencerelatedness between amino acid or nucleic acid sequences, as the casemay be, as determined by the match between strings of such sequences.“Similarity” between two amino acid sequences is determined by comparingthe amino acid sequence and its conserved amino acid substitutes of onepolypeptide to the sequence of a second polypeptide. “Identity” and“similarity” can be readily calculated by various methods, known tothose skilled in the art. In a preferred embodiment, sequence identityis determined by comparing the whole length of the sequences asidentified herein.

Preferred methods to determine identity are designed to give the largestmatch between the sequences tested. Methods to determine identity andsimilarity are codified in publicly available computer programs.Preferred computer program methods to determine identity and similaritybetween two sequences include, e.g., the BestFit, BLASTP, BLASTN, andFASTA (S. F. Altschul et al., J. Mol. Biol. 215:403-410 (1990), publiclyavailable from NCBI and other sources (BLAST Manual, S. Altschul et al.,NCBI NLM NIH Bethesda, Md. 20894). A most preferred algorithm used isEMBOSS (on the World Wide Web at ebi.ac.uk/emboss/align). Preferredparameters for amino acid sequences comparison using EMBOSS are gap open10.0, gap extend 0.5, Blosum 62 matrix. Preferred parameters for nucleicacid sequences comparison using EMBOSS are gap open 10.0, gap extend0.5, DNA full matrix (DNA identity matrix).

Optionally, in determining the degree of amino acid similarity, theskilled person may also take into account so-called “conservative” aminoacid substitutions, as will be clear to the skilled person. Conservativeamino acid substitutions refer to the interchangeability of residueshaving similar side chains. For example, a group of amino acids havingaliphatic side chains is glycine, alanine, valine, leucine, andisoleucine; a group of amino acids having aliphatic-hydroxyl side chainsis serine and threonine; a group of amino acids having amide-containingside chains is asparagine and glutamine; a group of amino acids havingaromatic side chains is phenylalanine, tyrosine, and tryptophan; a groupof amino acids having basic side chains is lysine, arginine, andhistidine; and a group of amino acids having sulphur-containing sidechains is cysteine and methionine. Preferred conservative amino acidssubstitution groups are: valine-leucine-isoleucine,phenylalanine-tyrosine, lysine-arginine, alanine-valine, andasparagine-glutamine. Substitutional variants of the amino acid sequencedisclosed herein are those in which at least one residue in thedisclosed sequences has been removed and a different residue inserted inits place. Preferably, the amino acid change is conservative. Preferredconservative substitutions for each of the naturally occurring aminoacids are as follows: Ala to ser; Arg to lys; Asn to gln or his; Asp toglu; Cys to ser or ala; Gln to asn; Glu to asp; Gly to pro; His to asnor gln; Ile to leu or val; Leu to ile or val; Lys to arg; gln or glu;Met to leu or ile; Phe to met, leu or tyr; Ser to thr; Thr to ser; Trpto tyr; Tyr to trp or phe; and Val to ile or leu.

It is well known to a person skilled in the art that 16s rRNA sequencescan be deposited online, for example, at GenBank (on the World Wide Webat ncbi.nlm.nih.gov/genbank/) and that they can be retrieved based ontheir unique accession number for use as reference 16S rRNA sequence inevaluation of sequence homology, such as, for example, described byEeckhaut et al. (2008). The GenBank accession numbers for the 16S rRNAsequences of seven bacterial species of the composition are listedbelow. These accession numbers can be used to retrieve the respective16S rRNA sequences from the World Wide Web at ncbi.nlm.nih.gov/genbank/for assessment of sequence homology.

GenBank accession number Species Strain (ncbi.nlm.nih.gov/genbank/)Roseburia hominis DSMZ 16839 AJ270482.2 (SEQ ID NO:1) Roseburiainulinivorans DSMZ 16841 AJ270473.3 (SEQ ID NO:2) Akkermansiamuciniphila DSMZ 22959 AY271254.1 (SEQ ID NO:3) Anaerostipes caccae DSMZ14662 AJ270487.2 (SEQ ID NO:4) Faecalibacterium DSMZ 17677 AJ270469.2(SEQ ID NO:5) prausnitzii Lactobacillus plantarum ZJ316 JN126052.1 (SEQID NO:6) Butyricicoccus LMG 24109 HH793440.1 (SEQ ID NO:7) pullicaecorum

The disclosure further relates to a composition as described hereinwherein the composition is a pharmaceutical composition formulatedeither as a rectally administrated form or an orally ingestible form.

In this regard, the disclosure further relates to a composition asdescribed herein wherein the orally ingestible form is a capsule,microcapsule, tablet, granule, powder, troche, pill, suspension orsyrup.

This disclosure further relates to a composition as described hereinthat is incorporated in a food, drink, food supplement or nutraceutical.

The disclosure thus relates to a composition as described herein that isused as food, food supplement or medicine for a human, a non-humandomestic or farmed land animal or an aquatic animal. The composition canthus be introduced in food, functional foods, food supplements,cosmetics, nutraceutical formulations, probiotic composition orpharmaceutical. A food is typically an edible material composedprimarily of one or more of the macronutrients protein, carbohydrate andfat. A food may also contain one or more micronutrients such as vitaminsor minerals. The term food as used herein also covers a beverage.Examples of foods in which the composition may be incorporated includesnack bars, cereals, buns, muffins, biscuits, cakes, pastries, processedvegetables, sweets, probiotic formulations including yogurts, beverages,plant oil-based liquids, animal fat-based liquids, frozen confectionsand cheeses. Preferred foods include yogurts, cheeses and other dairyproducts. Examples of beverages include soft beverages, syrups,squashes, dry drink mixes and nutritional beverages. A nutraceutical isa food ingredient, food supplement or food product that is considered toprovide a medical or health benefit, including the prevention andtreatment of disease. A functional food is a food that is typicallymarketed as providing a health benefit beyond that of supplying purenutrition to the consumer.

Also provided is a probiotic comprising a composition as discussedherein. A probiotic is typically a live supplement that can enhance theintestinal microbiota. Such probiotics may be given in particular tohumans but also to farm and domestic animals and to aquatic organisms.The probiotic may additionally comprise one or more acceptableexcipients or flavorings, which are suitable for ingestion by a human oranimal.

A composition of the disclosure may be used in the production ofpharmaceutical compositions. Thus, further provided is a pharmaceuticalcomposition comprising a composition of the disclosure and apharmaceutically acceptable excipient or carrier.

Compositions comprising compounds of the disclosure may be in diverseforms, for example, in the form of a tablet, capsule, or powder.Examples of excipients that may be present in such compositions includediluents (e.g., starch, cellulose derivatives or sugar derivatives), astabilizer (e.g., hygroscopic excipients such as silica ormaltodextrin), a lubricant (e.g., magnesium stearate), a buffer (e.g.,phosphate buffer), a binder, coating, preservative or suspension agent.Suitable excipients are well known to those skilled in the art.

This disclosure more specifically relates to a composition as describedherein wherein the composition comprises a total between 10⁵ and 10¹¹colony-forming units of bacteria of the disclosure.

In this document and in its claims, the verb “to comprise” and itsconjugations is used in its non-limiting sense to mean that itemsfollowing the word are included, but items not specifically mentionedare not excluded. In addition, the verb “to consist” may be replaced by“to consist essentially of” meaning that a composition as defined hereinmay comprise additional component(s) than the ones specificallyidentified, the additional component(s) not altering the uniquecharacteristic of the disclosure. In addition, reference to an elementby the indefinite article “a” or “an” does not exclude the possibilitythat more than one of the elements are present, unless the contextclearly requires that there be one and only one of the elements. Theindefinite article “a” or “an” thus usually means “at least one.”

All patent and literature references cited in the present specificationare hereby incorporated by reference in their entirety. The includedexamples are offered for illustrative purposes only, and are notintended to limit the scope of the disclosure in any way.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1: Schematic representation of a SHIME® unit that consists ofstomach, small intestine, and the three different colon regions. LiquidSHIME® nutritional medium and pancreatic juice enter the compartments,which simulate the stomach and small intestine, respectively. After adefined residence time in these sterile compartments, the suspensiongoes to three consecutive colon compartments, the ascending, transverse,and descending colon compartments, each characterized by distinct pHsand residence times. These compartments are inoculated with human fecalmicrobiota. All vessels are kept anaerobic by flushing the headspacewith N2, continuously stirred, and kept at 37° C.

FIG. 2: Butyrate production by 23 different compositions upon 24-hourincubation (top panel) and effect on the transepithelial electricalresistance (TEER) of Caco-2 cells cultured in the presence of THP1 cells(bottom panel). For the latter, samples collected from the 23incubations after 24 were sterile-filtered and added (1:5 v/v) for 24hours to the apical compartment of Caco-2 cells grown for fourteen dayson semipermeable inserts and placed on top of PMA-stimulatedTHP1-derived macrophages (co-cultures). Growth medium alone (DMEM) wasused as control. THP1 cells cultured in the presence of PMA for 48 hoursinduce damage on the Caco-2 cells as measured by a decrease in TEER inthe DMEM control. TEER values have been normalized to the valuesmeasured before co-culture (0 hour) and are expressed as percentage fromthe initial value. The coding of the different compositions was asfollows: MX-Y, in which X=number of isolates present in the compositionand Y=unique composition A, B, C, etc., with X isolates.

FIG. 3: Butyrate production upon 24-hour and 48-hour incubation inconditioned SHIME® nutritional medium by either the complete compositionof seven species or compositions of six species, in which each time oneof the seven original species was omitted. Results are presented as thepercentage of butyrate production detected in each incubation with acomposition of six species, as opposed to the composition consisting ofall seven species. Compositions are referred to as “Total” (all sevenspecies) or “Total−X,” with X being the species omitted from the totalcomposition. *: p<0.05 as compared to “Total” at 24 hours; #: p<0.05 ascompared to “Total” at 48 hours.

FIG. 4: Levels (mM) of butyrate, propionate and acetate produced by thecomposition throughout a five-day anaerobic incubation in conditionedSHIME® nutritional medium. The composition was either produced throughthe “Assembly” strategy (left panel) or the “Collaborome” strategy(right panel).

FIG. 5: Evolution of the levels (mM) of propionate (left panel) andbutyrate (right panel) over a fourteen-day time period in threeindependent production cycles of the composition through the“Collaborome” strategy. Upon initial growth in appropriate culturemedium, the strains of the composition were mixed, inoculated andcultured for fourteen days in triplicate in a SHIME® setup, consistingof a single colon region at a pH of 6.15-6.4.

FIG. 6: Evolution of SCFA levels expressed as mol % of acetate,propionate and butyrate over time, upon production of the compositionthrough the alternative “Collaborome” strategy. Upon initial growth inappropriate culture medium, the strains of the composition were mixed,inoculated and cultured for eight days in triplicate in singlefermenters operated in a fed-batch mode. At specific intervals of 16hours, 40% (v:v) of the growth medium was replaced with conditionedSHIME® nutritional medium.

FIG. 7: Production (mM) of acetate, propionate, butyrate and totalshort-chain fatty acids (SCFA) in 24-hour incubations with (i) sterilebasal medium (top panel), or sterile medium supplied with (ii)microbiota derived from a SHIME® colon region (middle panel) or (iii)fecal microbiota (lower panel). Different treatments with thecomposition, produced through the “Collaborome” strategy, were appliedranging from 0% to 4% and 20% of the total incubation volume.

FIG. 8: Evolution of levels (mM) of acetate (top panel), propionate(middle panel) and butyrate (lower panel) in an antibiotic recoveryexperiment in the M-SHIME®. Upon dysbiosis induction of theSHIME®-derived colon microbiota through administration of a cocktail ofantibiotics (40/40/10 mg/L of amoxicillin/ciprofloxacin/tetracycline,respectively), the dysbiosed microbiota was treated for five days withthe composition, produced either through the “Assembly” strategy or the“Collaborome” strategy (day 1=start of administration of thecomposition). The results are expressed as the delta of SCFA levels inthe SHIME® at each time point vs. the values before antibioticadministration.

FIG. 9: Levels (mM) of acetate (top panel), propionate (middle panel)and butyrate (lower panel) in an IBD-associated dysbiosis recoveryexperiment in the M-SHIME®. Three independent SHIME® colon vessels wereinoculated with fecal material from an Ulcerative Colitis patient.Simultaneously, a single dose of the composition, produced eitherthrough the “Assembly” strategy or the “Collaborome” strategy, was addedto a respective SHIME® colon vessel. A third experiment ran in parallelas control experiment without administration of the composition.Production of acetate, propionate and butyrate was followed one and twodays after administration of the composition.

FIG. 10: Evolution of levels (mol %) of acetate, propionate and butyratein an antibiotic recovery experiment in C57/BL6 mice. After a controlperiod in which the mice were fed a standard diet, gut microbiotadysbiosis was induced by adding clindamycin (250 mg/L) to the drinkingwater for five days. After this, the mice (n=10/group) were orallygavaged for five days with either saline solution (no bacterialintervention control; left panel), the composition, produced through the“Collaborome” strategy (middle panel) or the extended composition,produced through the “Collaborome” strategy (right panel). Mice fecalsamples obtained from the same intervention group were pooled and levelsof acetate, propionate and butyrate were quantified.

FIG. 11: Evolution of the Disease Activity Index (DAI) and weight changein a TNBS-induced colitis experiment in C57/BL6 mice. After a one-weekacclimatization period in which the mice were fed a standard diet, theexperiment was started. Each group (n=9/group) was treated for fiveconsecutive days by means of oral gavage. Preventive dosing of alltreatments started one day before the rectal administration of 2 mgTNBS/50% EtOH and lasted for four days after TNBS administration, beforemice were sacrificed. The following treatments were included:TNBS+saline solution (vehicle TNBS control); TNBS+composition, producedthrough the “Assembly” strategy, and TNBS+composition, produced throughthe “Collaborome” strategy. A conventional group (without TNBS treatmentbut treated with saline solution) was included as vehicle control.

FIG. 12: Evolution of the Disease Activity Index (DAI) in a DSS-inducedchronic colitis experiment in C57/BL6 mice. After a one-weekacclimatization period in which the mice were fed a standard diet, theexperiment was started. Each group (n=10/group) was treated three timesper week for eight consecutive weeks, by means of oral gavage.Preventive dosing of all treatments started one week before the firstDSS cycle. The first DSS cycle started on week 2 and included one weekof DSS administration (0.25% in drinking water) followed by two weeks ofrecovery. This first cycle was followed by an identical second DSScycle. The third DSS cycle consisted of one week of DSS administrationfollowed by one week of recovery, after which the animals weresacrificed. The following treatments were included: DSS+saline solution(vehicle DSS control); DSS+composition, produced through the“Collaborome” strategy. A conventional group (without DSS treatment buttreated with saline solution) was included as vehicle control.

DETAILED DESCRIPTION Examples Example 1: Establishment of a Composition1.1 Isolation of Bacteria for the Composition

A young, healthy donor with no prior exposure to antibiotic therapy wasselected to inoculate the SHIME® model. By controlling severaloperational parameters of the SHIME® model (FIG. 1, Van den Abbeele etal., 2010), one can enrich and select for networks of gut microbiotathat have a beneficial impact on human health such as microbiotainvolved in dietary fiber fermentation, bile acids metabolism, lactosedegradation, etc. The SHIME® setup was used for isolation of bacterialstrains with different functional properties, such as fiber degraders(e.g., Bifidobacteria, Bacteroides), fermentative (e.g., Escherichiacoli) or lactate producers (e.g., Lactobacilli, Pediococci andEnterococci), butyrate producers (e.g., Anaerostipes caccae,Butyricicoccus pullicaecorum, Faecalibacterium prausnitzii, Roseburiahominis, Roseburia inulinivorans, Clostridium butyricum) and propionateproducers (e.g., Bacteroides thetaiotaomicron, Bacteroides vulgatus,Roseburia inulinivorans, Akkermansia muciniphila). For this purpose,certain media were selected such as LAMVAB (lactobacilli; Hartemink etal. 1997), RB (bifidobacteria; Hartemink et al. 1996), Enterococcusmedium (Enterococci; Possemiers et al. 2004), TBX (Escherichia coli; LeBon et al. 2010), BBE (Bacteroides fragilis group; Livingston et al.1978), Mucin minimal medium (Akkermansia; Derrien et al. 2004), M2GSC(butyrate producers; Barcenilla et al. 2000) or lactate-containingminimal SHIME® medium (butyrate producers), succinate- andfucose-containing minimal SHIME® media (propionate producers),sulphate-enriched minimal media (sulphate reducers),arabinoxylan-containing minimal SHIME® medium and Blood agar plates(Prevotella). In addition to the SHIME®, bacteria were also isolateddirectly from a fresh fecal sample from a healthy donor, using the samestrategy.

In practice, ten-fold dilutions of samples collected from the coloniccompartments of the SHIME® or homogenized fecal samples were made andspread on agar plates with the specific medium composition as describedherein. Plates were incubated at 37° C., taking into account therespective growth conditions of the different bacterial groups. Uponincubation, approximately 30 colonies were picked up per bacterial groupand incubated in the respective liquid growth media under appropriateconditions. The short-chain fatty acid concentrations in the overnightcultures were analyzed using gas chromatography as described inPossemiers et al. (2004). Furthermore, a sample of the liquid cultureswas used for phylogenetic analysis. DNA was extracted as described inPossemiers et al. 2004 and the near-entire 16S rRNA sequences wereamplified for each isolate using the universal eubacterial primers fD1and rD1 (Weisburg et al. 1991). Upon purification, the DNA samples weresent out for sequencing. The obtained sequences were aligned withexisting sequences for identification of each isolate using the BLASTtoolbox (on the World Wide Web at blast.ncbi.nlm.nih.gov/Blast.cgi).

1.2 Design of the Composition

To combine different bacterial strains into actual functional microbialnetworks, the pure cultures isolated from the SHIME® reactor and fecalwere used (as described in Example 1.1). Additionally, pure cultureswere sourced from culture collections such as BCCM/LMG (World Wide Webat bccm.belspo.be) and DSMZ (World Wide Web at dsmz.de).

Short-chain fatty acids (SCFA) are the end products of dietary fibersfermentation by the intestinal microbiota and are known to exert severalbeneficial effects on host health. The main SCFA produced are acetate,butyrate and propionate in an approximate 60:20:20 molar ratio. Whereasacetate can be absorbed from the gut and used as energy substrate by thehost, butyrate acts as the main energy source for the gut epithelium andhas proven protective effects against inflammation and colon cancer.Propionate has similar local activity in the gut as compared tobutyrate, yet it is also transported to the liver where it was shown tohave positive cholesterol-lowering effects and effects on glycemiccontrol.

Considering the important and diverse physiological roles of SCFA,disruption of this gut microbial function (e.g., in gastrointestinaldisorders) can have a significant impact on host health. Consequently,in this example, a screening was performed to design a composition thatcan induce the highest total SCFA production and most interestingrelative SCFA production ratios. For the latter, butyrate was consideredthe most interesting among the different SCFA produced. Furthermore, theeffect of the different compositions on gut barrier integrity wasassessed by means of a co-culture of epithelial and immune cells.

In practice, a total 20 isolates with the most interesting fermentationprofiles, obtained from the isolation and selection round as describedin 1.1 (referred to as “Isolate-X”) or ordered from culture collections,were retrieved from their glycerol stocks and grown under theirrespective optimal growth conditions to obtain homogeneous suspensionsof the bacterial strains.

Ref Species Strain 1 Lactobacillus plantarum Isolate-1 2 Clostridiumbolteae Isolate-2 3 Desulfovibrio desulfuricans Isolate-3 4 Akkermansiamuciniphila Isolate-4 5 Coprococcus spp. Isolate-5 6 Roseburia hominisIsolate-6 7 Bacteroides Isolate-7 thetaiotaomicron 8 Clostridiumbutyricum Isolate-8 9 Anaerostripes caccae Isolate-9 10 BifidobacteriumIsolate-10 adolescentis 11 Faecalibacterium Isolate-11 prausnitzii 12Roseburia inulinivorans Isolate-12 13 Ruminococcus spp. Isolate-13 14Lactobacillus acidophilus Isolate-14 15 Enterococcus faecium Isolate-1516 Butyrivibrio fibrisolvens Isolate-16 17 Eubacterium limosum DSM20543Nissle 18 Escherichia coli 1917 19 Eubacterium rectale DSM17629 20Butyricicoccus Isolate-17 pullicaecorum

Isolates were combined in numbers ranging from 2 to 10 in a set of 98individual initial screening experiments. For each experiment,fermentation was started in sterile incubation bottles containingsterilized SHIME® nutritional medium adjusted to pH 6.8 withKH₂PO₄/K₂HPO₄ and flushed with nitrogen. Then, the sterilized medium wasinoculated with 10% (v/v) of mixed inoculum consisting of equal volumesof the selected species. Incubation bottles were flushed with nitrogento ensure anaerobic conditions and were incubated at 37° C. (90 rpm).Samples were analyzed after 24 hours for SCFA production. Compositionswith the highest butyrate production were then selected and further usedin the final experiment with 23 different sets of bacteria (referred toas MX-Y, in which X=number of isolates present in the composition andY=unique composition A, B, C, etc. with X isolates).

Identification number Composition M2-A 10, 12 M3-A 1, 9, 11 M4-A 1, 5,10, 11 M4-B 8, 10, 11, 17 M4-C 9, 10, 11, 13 M5-A 5, 8, 10, 13, 18 M5-B6, 9, 10, 11, 18 M6-A 5, 6, 9, 10, 12, 14 M6-B 2, 4, 8, 11, 13, 19 M6-C1, 4, 9, 11, 12, 17 M6-D 1, 6, 11, 13, 16, 20 M7-A 1, 3, 6, 9, 12, 16,20 M7-B 1, 4, 6, 9, 11, 12, 20 M7-C 6, 7, 13, 14, 16, 17, 20 M8-A 4, 5,6, 9, 10, 11, 13, 17 M8-B 4, 6, 7, 8, 11, 14, 16, 18 M8-C 1, 4, 8, 11,12, 15, 17, 20 M9-A 3, 6, 7, 11, 13, 14, 15, 17, 20 M9-B 3, 4, 6, 7, 14,15, 16, 18, 20 M9-C 2, 3, 5, 6, 7, 8, 12, 14, 20 M10-A 1, 3 ,4, 7, 8, 9,10, 12, 14, 15 M10-B 3, 4, 5, 7, 8, 9, 12, 14, 15, 16, 19 M10-C 2, 4, 6,8, 10, 11, 12, 13, 16, 18

These 23 combinations were again incubated as described before. After 24hours, samples were collected for SCFA analysis and for combination withthe co-culture model of Caco-2 and THP1 cells, as described inPossemiers et al. (2013). Endpoint of the latter experiment wasTrans-Epithelial Electrical Resistance (TEER) as measured for protectiveeffects toward gut barrier function.

FIG. 2 describes butyrate levels obtained upon 24-hour incubation of the23 different compositions as well as their effect on the TEER values.Strong variation was observed in both butyrate levels and effects on gutbarrier functioning and combinations with highest butyrate levels didnot necessarily induce highest protective effects on TEER levels, asshown by different ranking. Surprisingly, one composition of sevendifferent isolates (referred to as M7-B in FIG. 2) was ranked first onboth butyrate levels after 24 hours and especially on protective effectstoward gut barrier function. This composition contained six isolatesfrom the SHIME® and one culture obtained from a human fecal sample. 16SrRNA gene sequencing and comparison of the sequence with the NCBI BLASTdatabase revealed that M7-B was composed of novel SHIME® isolates ofLactobacillus plantarum, Faecalibacterium prausnitzii, Roseburiainulinivorans, Roseburia hominis, Akkermansia muciniphila andAnaerostipes caccae and of a novel fecal isolate of Butyricicoccuspullicaecorum. Interestingly, the novel isolates were all present in atleast one of the other compositions shown in FIG. 2, yet none of theother compositions reached the same effectivity with respect to butyrateproduction and protection of TEER values. This shows that the observedeffect is not related to one of the specific species present in thecomposition, but that only the specific combination of these sevenbacteria leads to the surprising positive results.

The seven novel isolates were deposited at the BCCM/LMG Bacteriacollection (Ghent Belgium), with accession numbers: Faecalibacteriumprausnitzii LMG P-29362, Butyricicoccus pullicaecorum LMG P-29360,Roseburia inulinivorans LMG P-29365, Roseburia hominis LMG P-29364,Akkermansia muciniphila LMG P-29361, Lactobacillus plantarum LMG P-29366and Anaerostipes caccae LMG P-29359.

As additional experimental evidence of the surprising synergy betweenthe seven isolates and the need for presence of each of the species, anexperiment was set up in which each time one of the isolates was removed(i.e., eliminated) from the original composition of seven isolates. Inpractice, fermentation was started again in sterile incubation bottlescontaining sterilized SHIME® nutritional medium adjusted to pH 6.8 withKH₂PO₄/K₂HPO₄ and flushed with nitrogen. Then, the sterilized medium wasinoculated with 10% (v/v) of mixed inoculum consisting of equal volumesof six of the seven isolates. The complete composition of seven isolatesacted as control, resulting in a total of eight parallel incubations.Incubation bottles were flushed with nitrogen to ensure anaerobicconditions and were incubated at 37° C. (90 rpm). Samples were analyzedafter 24 hours and 48 hours for butyrate production. As shown in FIG. 3,removal of only one species out of the original compositionsignificantly decreased butyrate production levels after 24 hours forall compositions of six species to below 80% of the butyrate productionof the original composition. Also, after 48 hours of incubation,butyrate levels were significantly lower for all compositions of sixspecies, with the exception of the composition excluding Roseburiahominis. This confirms that all isolates of the composition areessential to reach the full potential of the composition. As only thecomposition excluding Roseburia hominis still resulted in a similarfunctionality of the complete composition after 48 hours of incubation,one can also envisage, as second best, the use of the composition of sixspecies, consisting essentially of Faecalibacterium prausnitzii,Butyricicoccus pullicaecorum, Roseburia inulinivorans, Akkermansiamuciniphila, Lactobacillus plantarum and Anaerostipes caccae.

1.3 Production of the Composition

A composition consisting of the species Lactobacillus plantarum,Faecalibacterium prausnitzii, Butyricicoccus pullicaecorum, Roseburiainulinivorans, Roseburia hominis, Akkermansia muciniphila andAnaerostipes caccae is produced using three different strategies. Thesestrategies include either 1) growing the species of the compositionseparately, followed by mixing them together, 2) growing the species ofthe composition together in a multi-stage fermenter (i.e., the in vitroSHIME® model as described herein) and 3) growing the species of thecomposition together in a single-stage fermenter.

In the first strategy (the “assembly” strategy), the selected specieswere retrieved from their glycerol stocks and grown under theirrespective optimal growth conditions to obtain homogeneous suspensionsof the bacterial strains. To evaluate their functional activity, a mixedinoculum was created consisting of equal volumes of the selectedspecies. This inoculum was added at 10% (v/v) to sterile incubationbottles containing sterilized SHIME® nutritional medium adjusted to pH6.8 with KH₂PO₄/K₂HPO₄. Incubation bottles were flushed with nitrogen toensure anaerobic conditions and were incubated at 37° C. (90 rpm). Atspecific intervals of 16 hours, 40% (v:v) of the growth medium wasreplaced with conditioned SHIME® nutritional medium. Conditioned SHIME®nutritional medium was prepared by incubating 700 mL of normal SHIME®feed (pH 2) for one hour at 37° C., after which 300 mL of pancreaticjuice (pH 6.8)—supplemented with 25 g/L NaHCO₃, 23.6 g/L KH₂PO₄ and 4.7g/L K₂HPO₄—was added. Samples were analyzed over a period of five daysfor SCFA production (FIG. 4). Butyrate levels reached 7 mM upon 24 hoursincubation of the assembly and a maximum of 14 mM after five days.

In the second strategy (i.e., the “Collaborome” strategy or the strategy“wherein the bacteria are grown together in a dynamic simulator of thegastro-intestinal tract prior to administration”), the selected specieswere retrieved from their glycerol stocks and grown under theirrespective optimal growth conditions to obtain homogeneous suspensionsof the bacterial strains. Then, the strains were mixed and inoculated intriplicate in a SHIME® setup (Van den Abeele et al., 2010) consisting ofa single colon region at a pH of 6.15-6.4. A two-week adaptation periodwas implemented to create a functional collaborome composition. The needand relevance of such an adaptation period is clearly demonstrated bythe evolution of SCFA profiles during the cultivation of the compositionof selected species (FIG. 5). Initially, the composition requires timeto adapt to one another and to become active in converting the suppliedsubstrates to SCFA. However, four to six days after inoculation, theproduction of SCFA by the composition started to stabilize and highlevels of butyrate were measured. On the final day of incubation (day14), each of the triplicate incubations resulted in a highly similar,stable and strongly active functional composition with butyrate levelsreaching 19 mM.

When the stabilized Collaborome was frozen at −80° C. as glycerol stockand subsequently thawed for use as inoculum in the same way as for theassembly strategy, butyrate levels surprisingly increased faster andreached 25% higher levels under the same incubation conditions as forthe assembly of individual species (FIG. 4). Butyrate levels alreadyreached 12 mM upon 24-hour incubation of the assembly and a maximum of19 mM was already reached after two days.

In the third strategy, the production of the composition was undertakenusing an optimized single-stage fermenter approach, operated infed-batch mode (i.e., the alternative “Collaborome” strategy or thestrategy “wherein the bacteria are grown together in “a fermenter priorto administration”). The selected species were retrieved from theirglycerol stocks and grown under their respective optimal growthconditions to obtain homogeneous suspensions of the bacterial strains.Fermentation was started in sterile incubation bottles containingsterilized SHIME® feed adjusted to pH 6.8 with KH₂PO₄/K₂HPO₄ and flushedwith nitrogen. Then, the sterilized medium was inoculated with 10% (v/v)of mixed inoculum consisting of equal volumes of the selected species.Incubation bottles were flushed with nitrogen to ensure anaerobicconditions and were incubated at 37° C. (90 rpm). At specific intervalsof 16 hours, 40% (v:v) of the growth medium was replaced withconditioned SHIME® nutritional medium. Conditioned SHIME® nutritionalmedium was prepared by incubating 700 mL of normal SHIME® feed (pH 2)for one hour at 37° C., after which 300 mL of pancreatic juice (pH6.8)—supplemented with 25 g/L NaHCO₃, 23.6 g/L KH₂PO₄ and 4.7 g/LK₂HPO₄—was added.

As shown in FIG. 6, the total SCFA production and the ratio of SCFAproduced by the composition was stable after six replacement cycles.When re-inoculated in the same strategy as described before, thestabilized Collaborome led to a maximized SCFA production(acetate/propionate/butyrate ratio was around 14/12/74) two days earlieras compared to the same set of species in the assembly strategy and a25% higher butyrate production.

Example 2: In Vitro Experiments 2.1 Effect of Adding the FunctionalComposition to Complex Microbial Gut Communities

This experiment demonstrates that the functional composition is activewhen inoculated in a mixed microbial gut community, where there is astrong competition for colonic substrates with members of this complexintestinal community that is estimated to consist of 500 to 1000microbial species. To address this issue, an experiment was performed insmall incubation bottles using the composition, containing Lactobacillusplantarum, Faecalibacterium prausnitzii, Butyricicoccus pullicaecorum,Roseburia inulinivorans, Roseburia hominis, Akkermansia muciniphila andAnaerostipes caccae and produced through the Collaborome strategy fromExample 1.3. An increasing concentration of the pre-adapted composition(0%, 4% and 20%) was washed in PBS and added to three different media:

-   -   1) Sterile basal medium [2 g/L pepton, 2 g/L yeast extract, 2        mL/L TWEEN® 80, 10 μL/L, vitamin K1, 500 mg/L L-cysteine HCl,        100 mg/L NaCl, 40 mg/L K₂HPO₄, 40 mg/L KH₂PO₄, 10 mg/L        MgSO₄.7H₂O, 6.7 mg/L CaCl₂.2H₂O, 1.5 mg/L resazurin, 50 mg/L        hemin (50 mg/L)−pH 5.5]+starch 6 g/L;    -   2) Basal medium+20% fecal slurry (prepared as described in De        Boever et al., 2000);    -   3) Basal medium+20% SHIME® colon suspension, containing the        complete microbiota.

The increasing concentration of the pre-adapted butyrate-producingconsortium from 0% to 4% and 20% resulted in a proportional increase ofabsolute butyrate levels (FIG. 7). This was not only observed in sterilemedium, but also for media supplemented with a mixed microbiota derivedfrom both a fecal sample or a SHIME® colon region. This experiment thusdemonstrates that composition is not only active when present in anon-competing colonic environment, but that it also results in higherbutyrate levels when administered to a mixed microbiota where many gutmicrobes are competing for the same nutrients. Furthermore, not onlybutyrate production increased, but also propionate production stronglyincreased. The combination of these increases and the decrease ofacetate in the incubation stipulates that the composition can modulategeneral microbial fermentation profiles into a more health-beneficialprofile.

2.2 Efficiency of the Functional Composition to Restore the MetabolicFunctions of an Antibiotic-Induced Dysbiosed Gut Microbial Community

The use of antibiotics is believed to cause major disruptions of the gutmicrobiota community. It has been shown that a dysbiosed microbialcomposition is more susceptible to infections by pathogens. Furthermore,a number of gastrointestinal diseases have been correlated with adysbiosed microbial composition, such as inflammatory bowel diseases,underlining the importance of a healthy gut microbiome. Recovery of thetaxonomic composition and especially functionality after long-termantibiotic intake usually takes three months to reach the pre-treatmentstate, a healthy gut microbial community (Panda et al., 2014). Adecrease in the recovery time after exposure to antibiotic therapy couldthus reduce the risk of severe infections and promote host health ingeneral. In that respect, the observed functional activity of theselected composition could be a promising strategy to enhancerestoration of microbial communities upon antibiotics-induced dysbiosisand reduce infection risks.

In this example, antibiotics-induced dysbiosis was modeled in the invitro SHIME® model by dosing the appropriate antibiotics. The aim ofthis experiment was to evaluate the recovery of the typical “healthy”metabolite profiles in the simulated intestinal colon environments uponadministration of the functional composition. Furthermore, theexperiment aimed to differentiate the effectivity of the composition,when either produced through the “Assembly” strategy or the“Collaborome” strategy (see Example 1.3). The experiment was againperformed with the composition, containing Lactobacillus plantarum,Faecalibacterium prausnitzii, Butyricicoccus pullicaecorum, Roseburiainulinivorans, Roseburia hominis, Akkermansia muciniphila andAnaerostipes caccae. To better mimic the complete functionality profileof the intestinal microbiome, the composition was in this specificexperiment further supplemented with E. coli, Enterococcus faecium,Lactobacillus mucosae, Bifidobacterium adolescentis, Bifidobacteriumlongum, Bacteroides thetaiotaomicron and Bacteroides vulgatus.

In practice, SHIME® vessels (pH 6.15-6.40) were inoculated with fecalmaterial and allowed to stabilize for fourteen days (M-SHIME® setup—Vanden Abbeele et al., 2012). After a control period of two weeks, theSHIME®-derived colon microbiota was treated with a cocktail ofantibiotics (40/40/10 mg/L of amoxicillin/ciprofloxacin/tetracycline,respectively) to induce dysbiosis. One day later, the dysbiosedmicrobiota was treated for five days with the functional composition,produced either through the “Assembly” strategy or the “Collaborome”strategy. Endpoint of the study was to evaluate the recovery of thetypical “healthy” SCFA metabolite profiles in the simulated intestinalcolon environments. A control SHIME® vessel was included to simulatespontaneous recovery of the metabolic activity of the gut communityafter antibiotic exposure, without administration of the composition.The results are expressed as the delta of SCFA levels in the SHIME® ateach time point vs. the values before antibiotic administration (FIG.8).

Upon antibiotic treatment of the SHIME® vessels, a significant drop inacetate, propionate and butyrate production was observed. This findingconfirms the disruption of the gut microbial community. Recovery of themetabolite profile (in terms of SCFA production) to the pre-treatmentstate is shown in FIG. 8 as the evolution of acetate, propionate andbutyrate over a 5-day period. This shows that recovery of thefunctionality was slow in the control situation (no administration ofcomposition) and no full recovery could be observed for acetate andpropionate within five days. Interestingly, treatment with thecomposition resulted in a faster recovery as compared to the controlcondition for all three SCFA. Furthermore, while the composition of theAssembly strategy induced full recovery of propionate and butyrate afterfive days and three days, respectively, the composition of theCollaborome strategy induced a faster recovery as opposed to theAssembly strategy with full recovery of propionate and butyrate afterfour days and 2.5 days, respectively. Finally, the Collaborome strategyalso resulted in an increased final activity with increased propionateand butyrate levels as opposed to the Assembly strategy. These resultsemphasize the potential of the composition for the recovery ofantibiotic-mediated microbial dysbiosis. Moreover, this finding clearlydemonstrates that the preadaptation through the Collaborome strategyresults in a more efficient recovery of microbial SCFA production afterantibiotic exposure as compared to the Assembly strategy.

2.3 Efficiency of the Functional Composition to Restore the MetabolicFunctions of a Dysbiosed Gut Microbial Community in Inflammatory BowelDiseases

Inflammatory Bowel Diseases (IBD) have been associated with impairedhost-microbe interactions, which is related, at least partially, to astate of gut microbiota dysbiosis. The latter, for instance, includes alower abundance of butyryl CoA:acetate CoA transferase and propionatekinase (Vermeiren et al., FEMS 2011), which, in turn, negatively affectsthe production of a balanced SCFA production capacity. Given theimportant effects of SCFA on normal intestinal development andmaintenance, restoration of the microbiota composition and functionalityin terms of SCFA production can positively impact IBD-associatedsymptoms. In that respect, the observed functional activity of theselected composition could be a promising strategy to enhancerestoration of microbial communities in IBD dysbiosis as a basis forrestoration and maintenance of a healthy gut barrier.

In this example, IBD-associated dysbiosis was modeled in the in vitroM-SHIME® model, as described before (Vigsnaes et al. 2013). The aim ofthis experiment was to evaluate the recovery of the microbiota in termsof SCFA profiles in the simulated intestinal colon environment uponadministration of the functional composition. Furthermore, theexperiment aimed to differentiate the effectivity of the composition,when either produced through the “Assembly” strategy or the“Collaborome” strategy (see Example 1.3). The experiment was againperformed with the composition, containing Lactobacillus plantarum,Faecalibacterium prausnitzii, Butyricicoccus pullicaecorum, Roseburiainulinivorans, Roseburia hominis, Akkermansia muciniphila andAnaerostipes caccae.

In practice, SHIME® vessels (pH 6.15-6.40) were inoculated with fecalmaterial from an Ulcerative Colitis patient (M-SHIME® setup—Van denAbbeele et al., 2012). Simultaneously, a single dose of the functionalcomposition, produced either through the “Assembly” strategy or the“Collaborome” strategy, was added to the colon region to simulateadministration. A third experiment ran in parallel as control experimentwithout administration of the composition. Production of acetate,propionate and butyrate was followed one and two days afteradministration of the composition.

The results are presented in FIG. 9: administration of the composition,produced in the Assembly strategy, resulted in an increased SCFAproduction (mainly acetate and butyrate) on day 1, yet this effect wasno longer apparent on day 2. This indicates that the composition isfunctionally active in the IBD microbiome environment. Interestingly,the effect on propionate and butyrate production was much morepronounced upon administration of the composition of the Collaboromestrategy, with a four-fold and three-fold increase in propionate andbutyrate production, respectively, as opposed to the IBD control. Incontrast with the composition of the Assembly strategy, the effect wasstill pronounced on day 2 and coincided with a lower acetate production(indication of increased cross-feeding and, therefore, improvednetworking). These results emphasize the potential of the compositionfor the recovery of IBD-associated microbial dysbiosis. Moreover, thisfinding clearly demonstrates that the preadaptation through theCollaborome strategy results in a more efficient recovery of microbialSCFA production under IBD conditions as compared to the Assemblystrategy.

2.4 Efficiency of the Functional Composition to Inhibit Growth ofVegetative Clostridium difficile in an In Vitro Simulation Assay

In this example, a Clostridium difficile challenge test was performedaiming to evaluate whether the functional composition is not onlyfunctionally active under intestinal conditions, yet can also protectthe intestinal environment against infections. In such challenge test,the composition is challenged with vegetative Clostridium difficile(Cdif) cells to assess its capacity to inhibit growth of Cdif undersimulated gastro-intestinal conditions. Furthermore, the experimentaimed to differentiate the effectivity of the composition, when eitherproduced through the “Assembly” strategy or the “Collaborome” strategy(see Example 1.3). The experiment was again performed with thecomposition, containing Lactobacillus plantarum, Faecalibacteriumprausnitzii, Butyricicoccus pullicaecorum, Roseburia inulinivorans,Roseburia hominis, Akkermansia muciniphila and Anaerostipes caccae.

In practice, a glycerol stock of Clostridium difficile (LMG 21717^(T))was thawed and inoculated in a bottle containing Reinforced ClostridialMedium (RCM) broth that was flushed with nitrogen to ensure anaerobicconditions. The bottle was incubated in a shaking incubator (90 rpm) for24 hours and 10% of the grown culture was again inoculated in RCM broth.After 24 hours of growth, the homogenized C. difficile culture wasaliquoted (in triplicate) in bottles (10% v:v) containing:

-   -   1) Basal medium (blank);    -   2) Basal medium containing the composition of the Assembly        strategy;    -   3) Basal medium containing composition of the Collaborome        strategy;    -   4) Basal medium containing SHIME® colon suspension.

Bottles were incubated at 37° C. in a shaking incubator (90 rpm). Atregular time points, a sample was collected and immediately frozen at−80° C. before quantifying C. difficile by means of a qPCR assay basedon the detection and quantification of the triose phosphate isomerasegene. For this purpose, genomic DNA was extracted according to Boon etal. (2003). The amplification reaction included forward and reverseoligonucleotide: 5′-TATGGACTATGTTGTAATAGGAC-3′ (forward) (SEQ ID NO:8)and 5′-CATAATATTGGGTCTATTCCTAC-3′ (reverse) (SEQ ID NO:9). Absolutequantification of the PCR product was obtained by creating a standardcurve.

In this controlled in vitro simulation assay, growth of C. difficile wasobserved in the basal medium after 48 hours of incubation, confirmingthe validity of the blank in the in vitro simulation assay. The SHIME®colon suspension (as simulation of an actual fecal transplant) showedthe highest C. difficile growth inhibition after 48 hours of incubation(i.e., 58%). Interestingly, a similar result was obtained for thecomposition of the Collaborome strategy, showing approximately 53% of C.difficile growth inhibition. The lowest effect was observed when thecomposition of the Assembly strategy was added (i.e., 23% of growthinhibition). This experiment clearly demonstrates that C. difficile issignificantly inhibited in its growth by the composition and that thisinhibition is most pronounced in case of preadaptation of thecomposition through the Collaborome strategy.

2.5 Effect of the Functional Composition on Host Biomarkers of GutBarrier Functioning and Intestinal Immunity

Examples 2.1 to 2.3 showed that the composition is functionally activeunder complex intestinal conditions and can restore intestinalmetabolite profiles, with highest activity in the case of the productionthrough the Collaborome strategy. This may, in turn, beneficiallyinfluence the intestinal epithelium and thereby gut barrier functioningand local immunity.

To evaluate that possibility, this example describes the combination ofsamples collected from the previous experiments on an establishedco-culture cell model of enterocytes (Caco-2 cells) and macrophages(THP1) (Possemiers et al. 2013). In this model, stimulation of THP1cells with LPS results in increased production of pro-inflammatorycytokines, which, in turn, tends to disrupt the enterocyte layercreating a so-called “leaky gut” condition. The effect on the “leakygut” is measured by assessing the effect of the transepithelialelectrical resistance (TEER) [measurement for gut barrier efficiency]and inflammatory cytokine production, as compared to a controlcondition.

In practice, samples collected on day 1 from the M-SHIME® experimentfrom Example 2.3 were combined with the co-culture leaky gut model.

2.6 Impact of Variations in Strain Identity on Functional Activity ofthe Composition

To assess whether the surprising synergistic effect between the sevenisolates in the composition is strain specific or can also be reachedwith other strains of the same species, an additional experiment wasperformed. In this example, two different compositions are producedthrough the “Collaborome” strategy (see Example 1.3). While composition1 contains the specific isolates described in Example 1.2, composition 2is composed of strains from the same species obtained from culturecollections:

-   -   Composition 1: Faecalibacterium prausnitzii LMG P-29362,        Butyricicoccus pullicaecorum LMG P-29360, Roseburia        inulinivorans LMG P-29365, Roseburia hominis LMG P-29364,        Akkermansia muciniphila LMG P-29361, Lactobacillus plantarum LMG        P-29366 and Anaerostipes caccae LMG P-29359    -   Composition 2: Lactobacillus plantarum ZJ316, Faecalibacterium        prausnitzii (DSMZ 17677), Butyricicoccus pullicaecorum (LMG        24109), Roseburia inulinivorans (DSMZ 16841), Roseburia hominis        (DSMZ 16839), Akkermansia muciniphila (DSMZ 22959) and        Anaerostipes caccae (DSMZ 14662)

In practice, the selected species were retrieved from their glycerolstocks and grown under their respective optimal growth conditions toobtain homogeneous suspensions of the bacterial strains. Then, thestrains were mixed into Composition 1 and Composition 2, respectively,and each inoculated in triplicate in a SHIME® setup (Van den Abbeele etal., 2010) consisting of a single colon region at a pH of 6.15-6.4.Butyrate production profiles were followed up for a period of fourteendays.

Interestingly, the dynamics in butyrate production were highly similarfor both Compositions, with initial strong fluctuations, followed bystabilization of butyrate levels after approximately six days. At theend of the experiment (d14), butyrate levels for Composition 1 reached19.3 mM, while levels for Composition 2 were 18.8 mM. This shows thatthe synergistic effect observed in the composition from Example 1.2could be replicated by using different strains obtained from the samespecies.

Example 3: In Vivo Experiments 3.1 Mouse Model of Antibiotic-InducedGastrointestinal Microbiota Disruption

The goal of the experiment in this example was to assess whether thefunctional composition can also in an in vivo setting restore themetabolic capacity of the gut microbiome after antibiotic-induceddysbiosis.

In this example, the composition, containing Lactobacillus plantarum,Faecalibacterium prausnitzii, Butyricicoccus pullicaecorum, Roseburiainulinivorans, Roseburia hominis, Akkermansia muciniphila andAnaerostipes caccae, was used and produced via the “Collaborome”strategy of Example 1.3. Furthermore, to evaluate the need for morecomplete mimicking of the complete functionality profile of theintestinal microbiome, an extra experiment was performed in which thecomposition was further supplemented with Escherichia coli, Enterococcusfaecium, Lactobacillus mucosae, Bifidobacterium adolescentis,Bifidobacterium longum, Bacteroides thetaiotaomicron and Bacteroidesvulgatus (referred to as “extended composition”).

In practice, the “composition” and “extended composition” were preparedfresh according to the Collaborome strategy, washed twice in PBS (in ananaerobic chamber to ensure anaerobic conditions), concentrated in 100μL and administered to the mice via oral gavage as soon as possible.Mice (C57/BL6) of at least five weeks old were purchased, kept underpathogen-free conditions and fed a standard diet. Mouse experiments wereperformed in accordance with protocols approved by the Ethics Committeeof Animal Trials of Ghent University, Belgium. To induceantibiotic-induced dysbiosis, the antibiotic clindamycin was dosed tothe drinking water at a concentration of 250 mg/L. After five days ofantibiotic treatment, the stomach content of the mice was neutralizedwith NaHCO₃ after which the mice (ten mice per group) are orally gavagedfor five consecutive days with:

-   -   1) the composition in saline solution;    -   2) the extended composition in saline solution and    -   3) saline solution (control).

A conventional group (without antibiotic treatment but treated withsaline solution) is included as control to exclude variability arisingfrom the gavage procedure. During the experiment, fecal samples(approximately 100 mg/mouse) were collected and stored at −80° C. forfuture analyses.

The SCFA profiles, obtained from pooled mice fecal samples originatingfrom the same groups, demonstrate that five days of antibiotic treatmentsignificantly reduce butyrate and propionate production up to the extentthat only acetate remained (FIG. 10). As it is shown in FIG. 10,spontaneous recovery of the metabolic functions is slow and only startedabout five days (d10) after the last antibiotic treatment, although themolar ratios of the three major SCFA (acetate, propionate and butyrate)did not yet return to the pre-antibiotic state. When mice were, however,treated with either the composition or extended composition of theCollaborome strategy, recovery of butyrate metabolism already startedapproximately three days (d8) after antibiotic treatment. Furthermore,the metabolic activity of the mice treated with both compositions showedalmost complete recovery five days after the last dose of antibiotics(d10), with good production of both propionate and butyrate. Theextended composition contained a higher diversity of acetate andpropionate producers as compared to the composition, which is alsoreflected by the slightly different fermentation profile at d10 of theexperiment. In conclusion, this example provides an in vivo confirmationthat the functional composition is effective in obtaining a faster andmore potent recovery of intestinal metabolic profiles uponantibiotic-induced dysbiosis. Furthermore variations in the exactspecies combinations in the composition allows tuning the end resultinto specific metabolic profiles.

3.2 TNBS Mouse Model for Inflammation

The TNBS (2,4,6-trinitrobenzenesulfonic acid) model is a commonly usedmodel for colitis that mimics some of the features of Crohn's disease(Scheiffele et al. 2001), including weight loss, bloody diarrhea andintestinal wall thickening. On histopathology, TNBS causes patchytransmural inflammation of the gut with the formation of deep ulcers,classical features found in patients with CD. This makes the TNBS modela good candidate for in vivo evaluation of the capacity of thefunctional composition to prevent and/or restore damage to theintestinal mucosa in IBD and to assist in maintaining/developing ahealthy gut barrier.

In this example, the composition, containing Lactobacillus plantarum,Faecalibacterium prausnitzii, Butyricicoccus pullicaecorum, Roseburiainulinivorans, Roseburia hominis, Akkermansia muciniphila andAnaerostipes caccae was used to evaluate the beneficial effects uponevaluation in the TNBS model. Furthermore, the experiment aimed todifferentiate the effectivity of the composition, when either producedthrough the “Assembly” strategy or the “Collaborome” strategy (seeExample 1.3). Colitis was evoked in the animals by rectal instillationof TNBS, a mucosal sensitizing agent diluted in ethanol. Theadministration of ethanol is a prerequisite to break the colonic mucosalbarrier to allow penetration of TNBS into the lamina propria. TNBShaptenizes the localized colonic and gut microbial proteins to becomeimmunogenic, thereby triggering the host innate and adaptive immuneresponses.

In practice, eight- to ten-week-old male C57BL6/J mice were housed in atemperature-controlled room at 20° C. with a 12:12-hour light-darkcycle. The animals had free access to water and to a commercial chow.Mice were randomized among cages to avoid cage effects. After one weekof acclimatization, the experiment was started. Each group (n=9/group)was treated for five consecutive days by means of oral gavage.Preventive dosing of all treatments started one day before theadministration of 2 mg TNBS/50% EtOH rectally and lasted for four daysafter TNBS administration before mice were sacrificed. The followingtreatments were included:

-   -   1) TNBS+the composition of the Assembly strategy in saline        solution;    -   2) TNBS+the composition of the Collaborome strategy in saline        solution and    -   3) TNBS+saline solution (control).

A conventional group (without TNBS treatment but treated with salinesolution) is included as control to exclude variability arising from thegavage procedure. As study endpoint, Disease Activity was monitoreddaily (before the daily treatment) by measuring body weight, fecal bloodloss (ColoScreen) and general appearance.

The results of this example are presented in FIG. 11. No effects onweight nor Disease Activity were observed for the Vehicle (saline)control group without TNBS, while the control group that received TNBSshowed an immediate weight loss on dl of 8% and a strong increase inDisease Activity. Both weight loss and Disease activity were partiallyrestored by the end of the study. Interestingly, a potent protectiveeffect of the composition was observed on both weight loss and DiseaseActivity, yet the extent of this protective effect depended on theproduction strategy of the composition. While an initial mild protectionwas observed on dl for the Assembly strategy as shown to be lower weightloss and Disease Activity, this protective effect was no longer observedon the next study days. In contrast, the administration of thecomposition produced through the Collaborome strategy led to a potentpreventive effect toward weight loss and Disease Activity on dl, ascompared to the TNBS control, and a faster and complete restoration bythe end of the study, as shown by the return of the disease activity tothe level of the Vehicle control. In conclusion, this example providesan in vivo confirmation that the functional composition is effective inobtaining a stronger prevention of, and faster and more potent recoveryfrom, intestinal inflammation and Disease Activity upon TNBS-inducedcolitis induction. Moreover, this finding clearly demonstrates that thepreadaptation through the Collaborome strategy results in a moreefficient activity as compared to the Assembly strategy.

3.3 DSS Mouse Model for Inflammation

The chronic DSS model is a commonly used model for colitis that mimicssome of the features of Crohn's disease, including weight loss andbloody diarrhea. On histopathology, chronic DSS administration causesinflammation of the gut with typical architectural changes such as cryptdistortion, (sub)mucosal infiltration of inflammatory cells andfibrosis, features found in patients with CD. This makes the DSS model agood candidate for in vivo evaluation of the capacity of the functionalcomposition to prevent and/or restore damage to the intestinal mucosa inIBD and to assist in maintaining/developing a healthy gut barrier.

In this example, the composition, containing Lactobacillus plantarum,Faecalibacterium prausnitzii, Butyricicoccus pullicaecorum, Roseburiainulinivorans, Roseburia hominis, Akkermansia muciniphila andAnaerostipes caccae, and produced through the “Collaborome” strategy(see Example 1.3), is used to evaluate the beneficial effects uponevaluation in the chronic DSS model. Colitis is evoked in the animals byrepeated administration of DSS in the drinking water (0.25% challenge).The experiment is performed over a total of eight weeks, with threecycles of DSS administration and recovery.

In practice, six-week-old male C57BL6/J mice are housed in atemperature-controlled room at 20° C. with a 12:12-hour light-darkcycle. The animals have free access to water and to a commercial chow.Mice are randomized among cages to avoid cage effects. After one week ofacclimatization, the experiment is started. Each group (n=10/group) istreated three times per week for eight consecutive weeks, by means oforal gavage. Preventive dosing of all treatments starts one week beforethe first DSS cycle. The first DSS cycle starts on week 2 and includesone week of DSS administration (0.25% in drinking water) followed by twoweeks of recovery. This first cycle is followed by an identical secondDSS cycle. The third DSS cycle consists of one week of DSSadministration followed by one week of recovery, after which the animalsare sacrificed. The following treatments are included:

-   -   1) non-DSS control    -   2) DSS+the composition of the Collaborome strategy in saline        solution (three times/week) and    -   3) DSS+saline solution (DSS control).

As study endpoint, the Disease Activity Index (DAI) was monitored duringeach DSS cycle, three times per week (before the daily treatment) bymonitoring body weight, fecal blood loss (ColoScreen) and generalappearance. As shown in FIG. 12, no effects on DAI were observed for theVehicle (saline) control group without DSS, while the control group thatreceived DSS showed a strong increase in DAI at each administrationcycle. Interestingly, a potent protective effect (approximately 25%lower DAI at each cycle) of the composition was observed on DiseaseActivity. This further demonstrates that the functional composition iseffective in obtaining a strong protective effect from intestinalinflammation and Disease Activity upon DSS-induced colitis induction.

3.4 Mucositis Model

Mucositis is a clinical term used to describe damage to mucous membranesafter anticancer therapies. It occurs throughout the entiregastrointestinal tract (GT) (including the mouth) and genito-urinarytract, and to a lesser extent in other mucosal surfaces. Its severityand duration varies with the dose and the type of drug used. Theimportance of mucositis is that it limits the dose of chemotherapy. TheGI crypt epithelium is particularly vulnerable to chemotherapeutictoxicity, with symptoms including nausea and vomiting, abdominal pain,distension, and diarrhea due to direct effects of the cytotoxics on themucosa. The 5-fluorouracyl (5FU)-induced gut mucositis rat model wasestablished by Keefe et al. for assessment of the effects ofchemotherapy on the GI tract and it is now one of the most extensivelyused models to investigate chemotherapy-induced mucositis in rats (Keefe2004).

In this example, the composition, comprising Lactobacillus plantarum,Faecalibacterium prausnitzii, Butyricicoccus pullicaecorum, Roseburiainulinivorans, Akkermansia muciniphila and Anaerostipes caccae, was usedas the basis for the experiment and produced via the “Collaborome”strategy of Example 1.3. Mucositis is induced by means of a singleintraperitoneal dose of 5FU.

In practice, a total of 30 rats were randomly assigned to either acontrol or experimental group according to a specific time point. Allrats in the experimental groups received a single intraperitoneal doseof 5FU (150 mg 5FU/kg BW). Rats in the control groups received treatmentwith the solvent vehicle (dimethylsulphoxide). Subsequent toadministration of the chemotherapy drugs, study endpoints such asmortality, diarrhea, and general clinical condition were assessed fourtimes per 24-hour period. Subgroups of the rats were killed byexsanguination and cervical dislocation at 24, 48, and 72 hoursfollowing administration of the drug. Primary endpoints of interest wereevolution of weight, diarrhea and general wellbeing (sickness score).Secondary endpoints included histology of intestinal samples and stooland gut mucosal microbiota analysis.

To assess the effect of the composition on prevention or reducing theevaluated symptoms, part of the rats were administered for eightconsecutive days with the composition by means of oral gavage.Preventive dosing started five days before the administration of 5FU andlasted for three days after 5FU administration or until rats weresacrificed. Control animals did not receive the composition.

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What is claimed is:
 1. A method of treating a subject to reduce symptomsassociated with a gastro-intestinal disorder, the method comprising:administering to the subject a composition consisting of: bacteriabelonging to the species Faecalibacterium prausnitzii, Butyricicoccuspullicaecorum, Roseburia inulinivorans, Roseburia hominis, Akkermansiamuciniphila, Lactobacillus plantarum, and Anaerostipes caccae, andoptionally, a pharmaceutically acceptable excipient or excipient(s), soas to increase butyrate production or increase protective effectstowards gut barrier function in the subject and thus reduce symptomsassociated with the gastro-intestinal disorder in the subject.
 2. Themethod according to claim 1, wherein the gastro-intestinal disorder is adisruption of the barrier function of the gut, diarrhea, constipation,irritable bowel syndrome, inflammatory bowel disease, Crohn's disease,ulcerative colitis, coeliac disease, pouchitis, mucositis, an infectionof the gut, gut microbiota dysbiosis, and any combination thereof.
 3. Amethod of treating a subject to reduce symptoms associated with agastro-intestinal disorder, the method comprising: administering to thesubject a composition consisting of: bacteria belonging to the speciesFaecalibacterium prausnitzii, Butyricicocccus pullicaecorum, Roseburiainulinivorans, Akkermansia muciniphila, Lactobacillus plantarum, andAnaerostipes caccae, and optionally, a pharmaceutically acceptableexcipient or excipient(s), so as to increase butyrate production orincrease protective effects towards gut barrier function in the subjectand thus reduce symptoms associated with the gastro-intestinal disorderin the subject.
 4. The method according to claim 3, wherein thecomposition further includes one or more prebiotics.
 5. The methodaccording to claim 3, wherein the bacteria are grown together in afermenter prior to administration of the composition to the subject. 6.The method according to claim 5, wherein the fermenter is a dynamicsimulator of the gastrointestinal tract.
 7. The method according toclaim 3, wherein the gastro-intestinal disorder is a disruption of thebarrier function of the gut, diarrhea, constipation, irritable bowelsyndrome, inflammatory bowel disease, Crohn's disease, ulcerativecolitis, coeliac disease, pouchitis, mucositis, an infection of the gut,gut microbiota dysbiosis, and any combination thereof.
 8. The methodaccording to claim 3, wherein the composition further includes bacteriabelonging to the species Escherichia coli, Enterococcus faecium,Lactobacillus mucosae, Bifidobacterium adolescentis, Bifidobacteriumlongum, Bacteroides thetaiotaomicron and Bacteroides vulgatus.
 9. Themethod according to claim 3, wherein the bacteria are selected from thegroup consisting of Faecalibacterium prausnitzii LMG P-29362,Faecalibacterium prausnitzii DSMZ 17677, Butyricicoccus pullicaecorumLMG P-29360, Butyricicoccus pullicaecorum LMG24109, Roseburia 10inulinivorans LMG P-29365, Roseburia inulinivorans DSMZ 16841,Akkermansia muciniphila LMG P-29361, Akkermansia muciniphila DSMZ 22959,Lactobacillus plantarum LMG P-29366, Lactobacillus plantarum ZJ316,Anaerostipes caccae LMG P-29359, Anaerostipes caccae DSMZ 15 14662 andstrains showing at least 97% sequence identity to the 16S rRNA sequencesof at least one of the strains.
 10. A method of treating a subject toreduce symptoms associated with a gastro-intestinal disorder, the methodcomprising: administering to the subject a composition consisting of:bacteria belonging to the species Faecalibacterium prausnitzii,Butyricicoccus pullicaecorum, Roseburia inulinivorans, Akkermansiamuciniphila, Lactobacillus plantarum, Anaerostipes caccae, Escherichiacoli, Enterococcus faecium, Lactobacillus mucosae, Bifidobacteriumadolescentis, Bifidobacterium longum, Bacteroides thetaiotaomicron, andBacteroides vulgatus, and optionally, a pharmaceutically acceptableexcipient or excipient(s), so as to increase butyrate production orincrease protective effects towards gut barrier function in the subjectand thus reduce symptoms associated with the gastro-intestinal disorderin the subject.
 11. The method according to claim 5, wherein thebacteria, after having been grown together, have been frozen andsubsequently thawed for administration to the subject.
 12. A method oftreating a subject to reduce symptoms associated with agastro-intestinal disorder, the method comprising: administering to thesubject a composition consisting of: bacteria belonging to the speciesFaecalibacterium prausnitzii, Butyricicoccus pullicaecorum, Roseburiainulinivorans, Roseburia hominis, Akkermansia muciniphila, Lactobacillusplantarum, Anaerostipes caccae, Escherichia coli, Enterococcus faecium,Lactobacillus mucosae, Bifidobacterium adolescentis, Bifidobacteriumlongum, Bacteroides thetaiotaomicron, and Bacteroides vulgatus andoptionally, a pharmaceutically acceptable excipient or excipient(s), soas to increase butyrate production or increase protective effectstowards gut barrier function in the subject and thus reduce symptomsassociated with the gastro-intestinal disorder in the subject.
 13. Themethod according to claim 12, wherein the composition further includesone or more prebiotics.
 14. The method according to claim 12, whereinthe bacteria are grown together in a fermenter prior to administrationof the composition to the subject.
 15. The method according to claim 14,wherein the fermenter is a dynamic simulator of the gastrointestinaltract.
 16. The method according to claim 15, wherein the bacteria, afterhaving been grown together, have been frozen and subsequently thawed foradministration to the subject.
 17. The method according to claim 12,wherein the bacteria are selected from the group consisting ofFaecalibacterium prausnitzii LMG P-29362, Faecalibacterium prausnitziiDSMZ 17677, Butyricicoccus pullicaecorum LMG P-29360, Butyricicoccuspullicaecorum LMG24109, Roseburia 10 inulinivorans LMG P-29365,Roseburia inulinivorans DSMZ 16841, Roseburia hominis LMG P-29364,Roseburia hominis DSMZ 16839, Akkermansia muciniphila LMG P-29361,Akkermansia muciniphila DSMZ 22959, Lactobacillus plantarum LMG P-29366,Lactobacillus plantarum ZJ316, Anaerostipes caccae LMG P-29359,Anaerostipes caccae DSMZ 15 14662 and strains showing at least 97%sequence identity to the 16S rRNA sequences of at least one of thestrains.
 18. A reactor comprising: a composition consisting of from 5 to20 distinct bacterial members, wherein when the composition resides inthe reactor under standardized conditions representative of thegastrointestinal (GI) tract for a time of between 3 and 14 days, afunctionally stable collaborome is obtained, and wherein thestandardized conditions representative of the GI tract comprise: a pHrange of between 1.5 and 8; carbon sources; a retention time of between10 minutes and 200 hours; an oxygen availability of between 0 g/L and 8g/L; micronutrients; optional antibiotic(s); an optional bile saltconcentration of less than or equal to 20 mM; heavy metals; and hostfactors as immune molecules.
 19. The reactor of claim 18, wherein thecomposition consists of from 5 to 15 distinct bacterial members.
 20. Thereactor of claim 19, wherein the composition consists of from 6 to 14distinct bacterial members.
 21. The reactor of claim 18, wherein whenthe composition resides in the reactor under standardized conditionsrepresentative of the GI tract for a time of between 3 and 10 days, afunctionally stable collaborome is obtained.